Name: biomechanical testing of murine tendons
Uploaded: 2019-10-15T14:30:00
Description: Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

研究はNIH/NIAMS(R01 AR055580、R01 AR057836)によって支持された。

動物研究は、コロンビア大学機関動物ケアおよび使用委員会によって承認されました.この研究で使用されたマウスはC57BL/6Jの背景を有し、ジャクソン研究所(バーハーバー、ME、米国)から購入した。彼らは病原体を含まないバリア条件で収容され、食物と水のアドリビタムを提供された。
1. 骨を握るためのカスタムフィット3Dプリント器具の開発
<ol><li>骨画像取得と3D骨?...

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S., Connizzo, B. K., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+re-alignment+in+a+neonatal+developmental+mouse+supraspinatus+tendon+model.">Collagen fiber re-alignment in a neonatal developmental mouse supraspinatus tendon model.</a> Annals of Biomedical Engineering. 40, 1102-1110 (2012).</li><li>Cong, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+role+of+subacromial+impingement+in+rotator+cuff+tendinopathy:+Development+and+analysis+of+a+novel+murine+model.">Evaluating the role of subacromial impingement in rotator cuff tendinopathy: Development and analysis of a novel murine model.</a> Journal of Orthopaedic Research. 36, 2780-2788 (2018).</li><li>Thomopoulos, S., Birman, V., Genin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Interfaces+and+Attachments+in+Biology.">Structural Interfaces and Attachments in Biology.</a> Infection and Immunity. 35, (2013).</li><li>Boivin, G. P., 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=Biomechanical+properties+and+histology+of+db/db+diabetic+mouse+Achilles+tendon.">Biomechanical properties and histology of db/db diabetic mouse Achilles tendon.</a> Muscles, Ligaments and Tendons Journal. 4, 280-284 (2014).</li><li>Ansorge, H. L., Adams, S., Birk, D. E., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical,+Compositional,+and+Structural+Properties+of+the+Post-natal+Mouse+Achilles+Tendon.">Mechanical, Compositional, and Structural Properties of the Post-natal Mouse Achilles Tendon.</a> Annals of Biomedical Engineering. 39, 1904-1913 (2011).</li><li>Shu, C. C., Smith, M. M., Appleyard, R. C., Little, C. B., Melrose, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Achilles+and+tail+tendons+of+perlecan+exon+3+null+heparan+sulphate+deficient+mice+display+surprising+improvement+in+tendon+tensile+properties+and+altered+collagen+fibril+organisation+compared+to+C57BL/6+wild+type+mice.">Achilles and tail tendons of perlecan exon 3 null heparan sulphate deficient mice display surprising improvement in tendon tensile properties and altered collagen fibril organisation compared to C57BL/6 wild type mice.</a> PeerJ. 6, 5120 (2018).</li><li>Probst, 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=A+new+clamping+technique+for+biomechanical+testing+of+tendons+in+small+animals.">A new clamping technique for biomechanical testing of tendons in small animals.</a> Journal of Investigative Surgery. 13, 313-318 (2000).</li><li>Talan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body+temperature+of+C57BL/6J+mice+with+age.">Body temperature of C57BL/6J mice with age.</a> Experimental Gerontology. 19, 25-29 (1984).</li><li>Newton, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+testing+angle+on+the+biomechanical+properties+of+the+rat+supraspinatus+tendon.">The influence of testing angle on the biomechanical properties of the rat supraspinatus tendon.</a> Journal of Biomechanics. 49, 4159-4163 (2016).</li><li>Schwartz, A. G., Lipner, J. H., Pasteris, J. D., Genin, G. M., Thomopoulos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+loading+is+necessary+for+the+formation+of+a+functional+tendon+enthesis.">Muscle loading is necessary for the formation of a functional tendon enthesis.</a> Bone. 55, 44-51 (2014).</li><li>Gimbel, J. A., Van Kleunen, J. P., Williams, G. R., Thomopoulos, S., Soslowsky, L. 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I., 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=Ibuprofen+differentially+affects+supraspinatus+muscle+and+tendon+adaptations+to+exercise+in+a+rat+model.">Ibuprofen differentially affects supraspinatus muscle and tendon adaptations to exercise in a rat model.</a> American Journal of Sports Medicine. 44, 2237-2245 (2016).</li><li>Galasso, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+of+Life+and+Functional+Results+of+Arthroscopic+Partial+Repair+of+Irreparable+Rotator+Cuff+Tears.">Quality of Life and Functional Results of Arthroscopic Partial Repair of Irreparable Rotator Cuff Tears.</a> Arthroscopy - Journal of Arthroscopic and Related Surgery. 33, 261-268 (2017).</li><li>Sarver, D. 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マウス動物モデルは、腱障害を研究するために一般的に使用されますが、その機械的特性の特徴付けは困難であり、文献では珍しいです。このプロトコルの目的は、マウス腱の引張試験のための時間効率が良く、再現可能な方法を記述することです。新しい方法では、サンプルのテストに必要な時間が数時間から数分に短縮され、以前の方法で一般的な問題であった主要なグリップアーティ?...

腱障害は、老化、運動、および活性集団1、2、3の間で一般的で非常に一般的である。米国では、毎年1,640万人の結合組織損傷が報告され、すべての傷害関連の医師のオフィス訪問の3、5、6、7の30%を占めています。 8.最も一般的に影響を受ける部位には、回転子カフ、アキレス腱、膝蓋腱9が含まれる。抗炎症薬、リハビリテーション、外科的修復など、様々な非手術・手術治療が検討されているが、結果は依然として乏しく、機能への回帰が限られ、故障率が高い5 6.これらの貧弱な臨床結果は、腱症を理解し、新しい治療アプローチを開発しようとする基礎的および翻訳的研究を動機づけています。
引張生体力学的特性は、腱機能を定義する主要な定量的結果である。したがって、腱症および治療有効性の実験室的特徴付けは、腱引張特性の厳密な試験を含む必要があります。ラット、ヒツジ、イヌ、ウサギ10、11、12などの動物モデルから腱の生体力学的特性を決定する方法を数多くの研究が記載されている。しかし、主に引張試験のために小さな組織をつかむのが難しいため、マウス腱の生体力学的特性をテストした研究はほとんどありません。マウスモデルは、遺伝子操作、広範な試薬の選択肢、低コストなど、機械的に腱症を研究するための多くの利点を有するので、マウス組織を生体的にテストする正確で効率的な方法の開発が必要とされています。
腱の機械的特性を適切にテストするためには、組織を効果的につかむ必要があり、グリップ界面で滑ったり、実際に引き裂いたり、成長板を破砕したりする必要があります。多くの場合、特に短い腱の場合、骨は一方の端でつかまれ、腱はもう一方の端でつかまれる。骨は、典型的には、エポキシ樹脂13およびポリメチルメタクリレート14、15などの材料にそれらを埋め込むことによって確保される。腱は、多くの場合、サンドペーパーの2つの層の間に配置され、シアノクリレートで接着され、圧縮クランプ(断面が平らな場合)または凍結媒体(断面が大きい場合)15、16、17を使用して固定されています。.これらの方法は、生体力学的にマウス腱をテストするために適用されているが、試料のサイズが小さく、成長プレートのコンプライアンスが原因で問題が生じ、18を振動させない。例えば、マウスの頭の直径はわずか数ミリメートルで、骨のつかみにくくなります。具体的には、マウス上頭腱から骨までのサンプルの引張試験は、腱や腱のエンテシスではなく成長板で失敗することがよくあります。同様に、アキレス腱の生体力学的試験は困難である。アキレス腱は他のマウス腱よりも大きいが、カルカヌエウスは小さく、この骨のつかみ取りが困難である。骨を取り除き、その後に2つの腱の端をつかみます。しかし、これは腱と骨の付着のテストを妨げる。他のグループは、カスタムメイドの器具19、20、クランプ21による固定、自己硬化プラスチックセメント22で固定または円錐形スロット22を使用して、カルカインの骨をつかむ報告します。以前の方法は、再現性が低く、高いグリップ故障率、および退屈な準備要件によって制限されたままです。
今回の研究の目的は、マウス腱の引張生体力学的検査のための正確かつ効率的な方法を開発し、例として上脊椎およびアキレス腱に焦点を当てた。ネイティブ骨解剖学、固体モデリング、添加物製造からの3D再構成を組み合わせて、骨をつかむ新しい方法を開発しました。これらの器具は骨を効果的に確保し、成長板の故障を防ぎ、検体の調製時間を減らし、テストの再現性を高めた。この新しい方法は、ラットや他の動物の腱と同様に、他のマウス腱をテストするために容易に適応可能である。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Agarose</td>
			<td >Fisher Scientific</td>
			<td >BP160-100</td>
			<td >Dissovle 1g in 100 ml ultrapure water to make 1% agarose&#160;</td>
		</tr>
		<tr >
			<td >Bruker microCT&#160;</td>
			<td >Bruker BioSpin Corp</td>
			<td >Skyscan 1272&#160;</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >ElectroForce&#160;</td>
			<td >TA Instruments</td>
			<td >3200</td>
			<td>Testing platform</td>
		</tr>
		<tr >
			<td >Ethanol 200 Proof</td>
			<td >Fisher Scientific</td>
			<td >A4094</td>
			<td>Dilute to 70% and use as suggested in protocol</td>
		</tr>
		<tr >
			<td >Fixture to attach grips</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Kimwipes</td>
			<td >Kimberly-Clark</td>
			<td >&#160;S-8115</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >MicroCT CT-Analyser (Ctan)</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >MilliQ water (Ultrapure water)</td>
			<td>Millipore Sigma</td>
			<td>QGARD00R1 (or related purifier)</td>
			<td>100 ml&#160;</td>
		</tr>
		<tr >
			<td >Meshmixer</td>
			<td >Autodesk</td>
			<td >http://www.meshmixer.com/</td>
			<td >Free engineering software used by authors to refine mesh</td>
		</tr>
		<tr >
			<td >Objet EDEN 260VS&#160;</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Precision Prototyping</td>
		</tr>
		<tr >
			<td >Objet Studio</td>
			<td >Stratasys LTD</td>
			<td ></td>
			<td>Used by authors with 3D printer</td>
		</tr>
		<tr >
			<td >PBS - Phosphate-Buffered Saline</td>
			<td >ThermoFisher Scientific</td>
			<td >10010031</td>
			<td >2.5 L of 10% PBS&#160;</td>
		</tr>
		<tr >
			<td >S&#38;T Forceps</td>
			<td >Fine Science Tools</td>
			<td >00108-11</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Blade - #11</td>
			<td >Fine Science Tools</td>
			<td >10011-00</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Scalpel Handle - #3</td>
			<td >Fine Science Tools</td>
			<td >10003-12</td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >SkyScan 1272</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >Skyscan CT-Vox</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SkyScan NRecon</td>
			<td >Bruker BioSpin Corp</td>
			<td ></td>
			<td>Used by authors for visualizing and analyzing micro-CT scans&#160;</td>
		</tr>
		<tr >
			<td >SolidWorks CAD</td>
			<td >Dassault Syst&#232;mes</td>
			<td >SolidWorks Research Subsription</td>
			<td>Solid modeling computer-aided design used by authors</td>
		</tr>
		<tr >
			<td >SuperGlue</td>
			<td >Loctite</td>
			<td >234790</td>
			<td>As suggested in protocol</td>
		</tr>
		<tr >
			<td >Testing bath</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >Thin film grips&#160;</td>
			<td >Custom made</td>
			<td ></td>
			<td>Used by authors</td>
		</tr>
		<tr >
			<td >VeroWhitePlus</td>
			<td >Stratasys LTD</td>
			<td >NA</td>
			<td>3D printing material used by authors</td>
		</tr>
		<tr >
			<td >WinTest&#160;</td>
			<td >WinTest Software</td>
			<td ></td>
			<td>Used by authors to collect data</td>
		</tr>
	</tbody>
</table>

biomechanical testing of murine tendons

腱障害は一般的であり、すべての年齢の人々に影響を与え、しばしば衰弱している。抗炎症薬、リハビリテーション、外科的修復などの標準的な治療法は、しばしば失敗します。腱機能を定義し、新しい治療法の有効性を実証するためには、動物モデルからの腱の機械的特性を正確に決定する必要があります。マウス動物モデルは現在、腱障害を研究し、腱症の新しい治療法を評価するために広く使用されています。しかし、マウス腱の機械的特性を決定することは困難でした。本研究では、それぞれ上頭症腱とアキレス腱を機械的にテストするために、ヒューマラスとカルカヌスの解剖学に正確に一致する3Dプリント器具を含む腱機械試験用に新しいシステムが開発された。これらの器具は、ネイティブ骨解剖学、固体モデリング、および添加剤製造の3D再構成を使用して開発されました。この新しいアプローチにより、実際のグリップの失敗(腱ではなく成長プレートの故障など)が排除され、全体的な試験時間が減少し、再現性が向上しました。さらに、この新しい方法は、他の動物からの他のマウス腱や腱のテストに容易に適応可能である。

3Dプリントされた器具は、8週齢のマウス上脊椎間およびアキレス腱をテストするために使用された。すべての機械的にテストされたサンプルは、引張試験後のマイクロCTスキャン、目視検査、およびビデオ分析によって特徴づけられるように、封入で失敗しました。本研究室における上脊椎腱試験の前の方法と現在の方法の1対1の比較を図3に示す。前の方法28,29,30では、...

Watch this Scientific Journal Video about Biomechanical Testing of Murine Tendons at JoVE.com

Biomechanical Testing of Murine Tendons

このプロトコルは、カスタムフィット3Dプリント器具の使用を通じて、マウス腱の効率的で再現性の高い引張生体力学的試験方法について説明します。

マウス腱の生体力学的試験

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

このプロトコルは、再利用可能なカスタムフィット3Dプリントフィクスチャを通じて、マウス腱の生体力学的検査のための効率的で再現可能な方法を記述しています。新しい方法では、サンプルのテストに必要な時間を時間から分に短縮し、以前のメソッドで一般的な問題であった主要なグリップアーティファクトを排除します。この方法は、上スピナトゥス腱およびアキレス腱に限定されない。それはより大きい動物からの他のマウス腱および腱をテストするために容易に合わせることができる。このアプローチの再現性と効率を確保するために、ここで説明されていないフィクスチャに対して、設計のプロトタイプ作成と検証の複数のサイクルが必要になる場合があります。まず、アガロースゲル中の骨全体のマイクロコンピュータ断層撮影を行う。断面イメージへの再構築後に、[ファイルを開く] コマンドを選択してファイル データセットを開きます。ダイアログファイルの環境設定を開き、[詳細設定]タブを選択します。アダプティブ レンダリング アルゴリズムを使用して、サーフェスの詳細を滑らかにする 3D モデルを構築します。オブジェクトの境界線を見つけるために使用される隣接点までの距離をピクセル単位で定義する、地域性パラメータとして 10 を使用します。許容値を 0.1 に最小化して、ファイル サイズを小さくします。対象のボリュームを指定するには、選択した VOI 範囲の上部と下部に設定する 2 つのイメージを手動で選択し、2 番目のページの [対象地域] に移動します。単一の断面イメージ上の対象領域を手動で選択します。10 ~15 枚の断面画像ごとに ROI を繰り返します。次に、3 番目のページである [バイナリ選択] に移動します。[ヒストグラム] メニューの [データセットから] をクリックして、データセットのすべての画像から明るさのヒストグラム分布を表示します。また、[ヒストグラム] メニューの [3D モデル ファイルの作成] メニューをクリックします。ボーンの 3D モデルを STL ファイル形式で保存します。Meshmixer ソフトウェアでメッシュをインポートして[すべて]を選択して編集します。トリカウントを減らし、変更を受け入れます。STL形式で新しく縮小されたファイルを保存し直すAs.To上副脊髄腱骨を設計するを選択し、固体モデリングコンピュータ支援設計プログラムを使用して上腕骨をつかむフィクスチャのカスタムフィットモデルを作成します。ソリッドモデリングプログラムで上腕骨のSTLフォーマットファイルを開き、SLDPRT形式でパーツファイルとして保存します。次に、Part ファイルを開き、サジタル、コロナ、横など、3 つの解剖学的に関連する平面を手動で作成します。[ファイル新規作成]をクリックして、ソリッド ブロック コンポーネント パーツを作成します。[ファイル新規作成]をクリックすると、ソリッド ブロックと右または左の上腕骨の 2 つの構成部品を含むアセンブリ モデルが作成されます。ブロック内のボーンの方向を定義して、腱とボーンの間の角度を 180 度にします。ボーンボリューム全体がブロック内に収まることを確認します。[アセンブリ]ウィンドウでブロックを選択し、[アセンブリ]ツールバーの[コンポーネントを編集]をクリックします。挿入、フィーチャー、キャビティをクリックします。[均等スケーリング]を選択し、すべての方向にスケーリングする値として0%を入力します。ボーン部品を省略し、アセンブリを部品として保存します。キャビティパーツで円柱を開きます。スケッチを作成し、上腕頭の上に0.5ミリメートルだけ残すことを確認してください。[フィーチャ]から、[押し出し切り]を選択します。矢状平面に沿ってアセンブリを切断し、骨を前部および後部に収める2つの対称的なコンポーネントを作成します。矢状平面に沿ってアセンブリ部品をカットして、骨を前部および後方にフィットする 2 つの対称的なコンポーネントを作成します。成長プレートの故障を防ぐために上腕頭を確保し、テスト中に上腕骨がモデルから外れないようにする密閉的なクリアランスを定義することが重要です。原稿に記載されているとおりに進み、後部成分と前部成分を仕上げ。両方のコンポーネントを個別の部品ファイルとして保存します。今度は、[パターンを挿入]、[ミラー]を押して[ミラー]を選択し、反対側の四肢のフィクスチャの各コンポーネントに対して 3D ミラー モデルを作成します。正面を鏡面平野として選択します。ミラーするボディとしてパーツを選択します。ミラー平面を選択し、すべての材料を含むスケッチを作成します。フィーチャーから、元の部品を除去し、ミラー化された部品のみを保持するには、[押し出し切り]を選択します。部品の下部をクリックし、スケッチを作成する平面として使用します。[スケッチ文字]をクリックして L または R を追加します。3D プリントの準備として、すべてのフィクスチャパーツを STL 標準ファイル形式で保存します。上腕脊髄性筋腱、上腕骨の骨標本を解剖するために、最初に安楽死させたマウスを起こしやすい位置に位置し、前足の肘の上から肩に向かって皮膚の切開を行う。肩の筋肉が見えるように、鈍い解剖で慎重に皮膚を除去するために鉗子を使用してください。骨が露出するまで上腕骨を取り巻く組織を取り除きます。鉗子で上腕骨を保持し、慎重にコラ血科のアーチを露出するために三角筋とトラペジウス筋肉を除去します。ACジョイントを識別し、慎重にメスのブレードでアクロミオンから鎖骨を分離します。上頭蓋腱とその骨の付着を損傷しないように注意して、メスの刃を使用して肩甲骨付着から筋肉を取り除きます。グレノイドから上腕頭を取り外し、関節カプセルと眼圧下垂体と短靭腱を裂きます。肘関節を非連結して、上腕骨と尺骨と半径を分離する。上腕上腕脊髄性腱筋標本を分離し、上腕骨および上腕骨の頭部の余分な軟部組織をきれいにする。アキレス腱を解剖するために、踵骨を、安楽死させたマウスを起こしやすい位置に位置する。アキレス腱とその骨の付着を損傷しないように注意して、足首と膝関節の周りの筋肉が露出するように鈍い解剖で皮膚を取り除きます。メスの刃を使用して、アキレス腱-カルカニュースの付着から始めて、慎重にその近位の付着から胃頭筋を取り外します。様々な隣接する骨から慎重にカルカニューを除去し、アキレス腱-カルカニューサンプルを分離し、余分な軟組織をきれいにします。腱の断面積を決定するには、骨を逆さまに挿入して、アガロースゲルの骨と腱と筋肉を外側に含むアガロースゲルで満たされたクライオチューブに標本を吊り下げる。マイクロコンピュータ断層撮影を使用してスキャンした後、メスの刃を使用して腱から筋肉を静かに取り除きます。3D プリントされたフィクスチャにボーンを挿入し、テスト グリッドにアタッチします。折りたたまれた薄いティッシュペーパーの間に腱を挿入して接着し、薄膜グリップを使用して締め付けます。サンプルとグリップを37°CのPBSの試験浴場に挿入し、機械的なテストを行います。本研究では、再利用可能な3Dプリントされたフィクスチャを使用して骨を握り、検体の調製時間を数時間から数分に短縮した。現在の方法を用いた上垂脊髄腱の引張試験のための代表的な負荷変形曲線は、成長板の故障などの主要な把持アーティファクトを排除した。上スピナトゥス腱とアキレス腱の機械的特性は、P値が0.05未満の非対Tテストに基づいてセックスの有意な効果を示す。マイクロCTは、アキレス腱の長さに沿った上脊髄腱の長さに沿った断面積の測定に成功した。各解剖学的部位には、効果的なグリップに必要な特定の設計基準があることを覚えておくことが重要です。したがって、各器具の設計は、それに応じて適合する必要があります。故障までの引張試験に加えて、腱疲労および粘性特性に関する情報を提供する周期的な負荷試験に使用することができる。マウス腱の機械的特性を特徴付けるのは、これらの小さな組織をつかむのが難しいこと、退屈で時間のかかる標本調製方法、および成長プレート骨折を含む多くの理由から、文献では珍しい。このプロトコルは、実際のグリップの失敗を排除し、1日にテストできる標本の数を3倍にしたマウス腱をテストするための時間効率と再現性の高い方法を提示します。

Research

JoVE Journal

Bioengineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

生体力学的解析と組織工学のための細胞由来の組織のリングを製作する監督セルラーセルフアセンブリ

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

生物工学

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

使用プラ​​センタアクロス異物およびナノ材料の輸送速度の決定<em&gt; ex vivoで</em&gt;ヒト胎盤灌流モデル

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

インデントと引張試験を用いたヒト軟部組織の生体力学的特性評価

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

実験および有限要素議定書は、関節軟骨全体で中性および荷電溶質の輸送を調査します

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during an impact are important factors in protecting the helmet wearer from impact-induced injury. This manuscript aims to investigate impact-induced injury mechanisms in different helmet fit scenarios through analysis of simulated helmeted impacts with an anthropometric test device (ATD), an array of headform acceleration transducers and neck force/moment transducers, a dual high speed camera system, and helmet-fit force sensors developed in our research group based on Bragg gratings in optical fiber. To simulate impacts, an instrumented headform and flexible neck fall along a linear guide rail onto an anvil. The test bed allows simulation of head impact at speeds up to 8.3 m/s, onto impact surfaces that are both flat and angled. The headform is fit with a crash helmet and several fit scenarios can be simulated by making context specific adjustments to the helmet position index and/or helmet size. To quantify helmet retention, the movement of the helmet on the head is quantified using post-hoc image analysis. To quantify head and neck injury potential, biomechanical measures based on headform acceleration and neck force/moment are measured. These biomechanical measures, through comparison with established human tolerance curves, can estimate the risk of severe life threatening and/or mild diffuse brain injury and osteoligamentous neck injury. To our knowledge, the presented test-bed is the first developed specifically to assess biomechanical effects on head and neck injury relative to helmet fit and retention.

Using an anthropometric head and neck, optical fiber-based fit force transducers, an array of head acceleration and neck force/moment transducers, and a dual high speed camera system, we present a test bed to study helmet retention and effects on biomechanical measures of head and neck injury secondary to head impact.

ヘルメットのフィットと保持と頭とシミュレートされた衝撃で頸部損傷の生体力学的措置を検討するテスト ベッド

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

新生児子豚における腕神経叢における生体内生体力学試験の方法

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

圧力筋グラフを用したマウス生殖系の二軸基底音と受動的試験

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

The Quantification of Injectability by Mechanical Testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

機械的試験による注入性の定量化

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

冠動脈動脈硬化の光コヘレンス断層撮影法による生体力学的流体構造相互作用解析

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

大動脈壁における生体力学的ひずみを再現するためのヒト大動脈平滑筋細胞器官チップモデルの構築

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...