我々は、我々の研究の支援とコラボレーションのための博士ジョージ·コモンズ博士とディーンVistnesに感謝したいと思います。セントジョセフマーシー病院GMEによってサポートされているこの作品は、歯科および頭蓋顔面研究助成1 R21はDE019274-01、R01EB009689およびRC2 DE020771-02、MTL博士ヒョンに小児再生医療のためのオーク財団とHagey研究所の国立研究所によってサポートされています。

1。細胞分離および増殖<ol><li>すべての患者の同意と実験プロトコルはスタンフォード大学の治験審査委員会（プロトコル＃2188と＃9999）によって検討され、承認された。 </li><li>一般的/局所麻酔下での選択科目lipoaspiration手続きからヒト皮下脂肪組織を取得します。 </li><li>吸引脂肪の2つの層（ 図1A）があるでしょう。上清は、処理細胞物質の大半が含まれています。底層は、主に生理食塩水が注入される。 脂肪由来間質細胞は、いずれかの層から採取したが、収量は清からはるかに大きいことができます。 </li><li>間質血管画分（SVF）を分離し、1Xの等量で広範囲に脂肪吸引を洗うベタジンずに1×PBS（x1）と等量のに続いて2.5％ベタジン（x2）を含む生理食塩水（PBS）、リン酸緩衝。沈殿に洗浄を可能にします。 </li><li>滅菌TEを維持するように注意してください処理されたヒト組織による汚染が起こることができるようにプロセス全体chnique。 </li><li>底層を吸引除去し、各洗浄後に破棄します。 </li><li> 50ミリリットルBDファルコンコニカルチューブに脂肪組織のアリコート15ミリリットル。 </li><li> 37℃ハンクス液でフィルタリング0.075%タイプIIコラゲナーゼの等量（15ml）中（HBSS）℃の水浴中で60分間一定の攪拌下（約180 /分を振る）と脂肪組織を消化 - 各チューブに15分ごとに換気を行ってください。 </li><li> 15ミリリットルPBSで4℃で5分間1,000 rpmで10％（ウシ胎児血清）FBS、遠心分離を含有する酵素活性を中和℃で高密度SVFペレットを得た。ペレットは、脂肪由来の間質細胞と赤血球のミックスになります。 </li><li>ペレットを中断することなく、上清を捨てる。 </li><li>伝統的な増殖培地（ダルベッコ改変イーグル培地[DMEM] / 10％FBS / 1％ペニシリン - ストレプトマイシン溶液）10ml中にペレットを再懸濁します。 </li><li>フィルタサスペンション<html>細胞残渣を除去するために100μmナイロンセルストレーナーを通してシオン。 </li><li> 1クリーン50mlコニカルチューブに3を兼ね備えています。 4℃で5分間1,000 rpmで遠心する</li><li>ペレットを中断することなく、上清を捨てる。 </li><li>伝統的な増殖培地5mlにペレットを再懸濁し</li><li> 1 10cmプレートまたは1 15cmのプレートのための5つの50ミリリットルconicalsごとに2つの50ミリリットルconicalsを兼ね備えています。 </li><li> 37°C/21％O 2、5％CO 2で一晩初代培養を確立します。 </li><li>一晩インキュベートした後、残留非付着赤血球を除去するためにPBSでプレートを洗浄する。得られた細胞集団は、脂肪由来の間質細胞である。 </li><li> 37°C/21％O 2、5％CO増殖培地中2で自発的分化を防止するためのサブコンフルエントレベルで細胞を維持。細胞は一般的に50％コンフルエントで播種したときに定期的な増殖培地で3〜4日ごとに分割する必要があります。 </li></ol>ve_title &quot;&gt; 2。足場の準備<ol><li>足場は、カリフォルニア大学ロサンゼルス校博士ミンリーによって作られた - 歯学部。 </li><li> PLGAの足場は、溶媒キャストおよび粒状浸出法によりポリ85/15（乳酸-co-グリコール酸）（固有粘度= 0.61（dl / g）で、バーミンガム·ポリマーズ）から作製した。 </li><li> PLGA /クロロホルムソリューションは92％の気孔率を（体積分率）を得るために、200から300μmの直径のスクロースと混合し、テフロン製の型に薄いシート状に圧縮した。 </li><li>一晩凍結乾燥した後、足場がスクロースを溶解させるために二重蒸留（DD）のH 2 O 3の変更に浸漬し、ゆっくりと精密チップ·スパチュラでテフロン板から削除されます。 </li><li>微粒子浸出した後、すべての足場は蒸留H 2 Oの3回のすすぎに続いて、30分ごとに50％、60％、70％エタノールに浸漬して消毒した</li><li>すべての足場は、層流フード下で乾燥した。 </li><li>足場製作、sの後にcaffoldsは、アパタイトを被覆した。 <ol class="ualpha"><li> SBF（擬似体液）溶液は、ヒト血漿のそれの5倍であったイオン濃度で調製した。すべてのソリューションは0.22μmのPES膜（ナルゲン）を通して濾過滅菌だった。直ちにコーティングプロセスの前に、乾燥したPLGAの足場は濡れおよびコーティングの均一性を向上させるためにグロー放電、アルゴンプラズマエッチング（Harrickサイエンティフィック）に供した。 </li><li>エッチングされたPLGA足場はその後37℃でインキュベートしたSBF°のMg 2に続いて12時間水ジャケットインキュベーターの中でC、+とHCO 3 - 37℃でさらに12時間のための無料のSBF 2℃穏やかに撹拌しながら。 </li><li>コーティングされたPLGAの足場は、層流フード内で乾燥させ、70％エタノールで消毒し、過剰の塩化ナトリウム溶液を洗い流すために、無菌ddH 2 Oで穏やかに洗浄した。 </li><li>アパタイトコーティングの完全性は、走査型電子顕微鏡（SEM）を用いて解析した。アパタイトでコーティングされたSCAffoldsは、SEMスタブ（テッド·ペラ）に搭載され、導電性を向上させるために炭素で被覆した。二次電子モードは、SEM（FEI /フィリップスXL-30）観察中に適用された。エネルギー分散X線スペクトルは、アパタイト構造の元素組成を確認するために得られた。 </li></ol></li></ol> 3。細胞播種<ol><li>サブ合流レベルに到達すると、PBS（×2）で細胞を洗浄し、トリプシン処理。 </li><li>シードするために定量化するために細胞数を数える</li><li> 1.5×10 5個の細胞を持つプレカット4.0 mm径の足場の上にシード。 <ol class="ualpha"><li> 96ウェルプレートのウェルに置き、個々の足場</li><li> メディアの20μlあたり約1.5×10 5細胞の濃度との定期的な増殖培地で再懸濁細胞 </li><li>足場と30分間、細胞培養インキュベーターに場所に直接ピペットで20μlの。 </li><li> 30分後、メディアを200μl加え、細胞がインキュベートすることができます手術前に一晩で足場</li><li>それは、大半が足場上に添付するのを確実にするでしょう十分な細胞数を播種すると足場の上に細胞の接着性を確保する必要はありません。これは、生物発光と組織学的分析を用いた in vivo で弊社ラボで検証されています。 </li></ol></li></ol> 4。頭蓋冠の欠陥の生成と生体内注入時の <ol><li>麻酔カクテルを持つ成人（60日齢）のCD-1ヌードマウスを麻酔。 （ケタミン/キシラジン/アセプロマジン）50％濃度（0.9％NaClで1:1希釈）で、または機関プロトコルあたりの推奨麻酔薬レジメンごと。 hASCsが免疫反応を引き起こすことはありませんので、これらのマウスは、無胸腺です。 <ol class="lalpha"><li>投与量：塩酸ケタミン（80 mg / kg）を、キシラジン（2.5 mg / kg）をアセプロマジン（2.5 mg / kg）を</li><li>効果の持続時間：約30分</li></ol></li><li>外科的に動物をドレープと手術を殺菌ベタジンおよびアルコール（×3）と敷地前に右頭頂骨を露出させるために動物の頭皮の正中矢状切開を作るために軟膏を使用して、マウスの目を覆う。鈍スクレイピング（ 図1B）と右頭頂骨から頭蓋骨膜を取り除きます。 </li><li>無菌性ダイヤモンド被覆の穿孔ドリルビットを使用して、右の非縫合関連する頭頂骨における一方的な4 mmの全層欠損を作成します。 細心の注意を払ってマウス頭蓋冠の厚さに応じて基本となる硬膜（図1B）を乱さないように注意しなければなりません &lt;0.3ミリメートルです。 </li><li>注入前に、培地由来成長因子の移動を防止するために滅菌PBSで足場をすすいでください。 </li><li>欠損部に足場を置きます。 </li><li>最後に、皮膚が閉じ縫合および確立術後のプロトコルごとに動物を監視します。 </li><li>術後、 例えば 、必要に応じて動物の世話·プロトコル監督として確立鎮痛薬を使用してください。ブプレノルフィン0.1 mgの/ Kグラム。 </li></ol> 5。類骨形成の定量<ol><li> MicroCTをマウスで行った後、3次元画像は前述の1としてMicroViewとソフトウェアを使用して再建された。 </li><li> Adobe Photoshopを使用して、画像は標準の高さ大きさとされた。 </li><li>魔法の杖の機能を使用すると、ピクセルは頭蓋冠の欠陥を測定した。 </li><li>欠陥の割合治癒はその後のCTスキャン頭蓋冠欠損面積を測定し、画素数を決定し、元の欠陥の画素数で割って算出された</li><li> MicroCTが使用できない場合はそのため、代替は、Adobe Photoshopを使用し、組織学のために決定された時点で頭蓋を収穫しています。 </li><li>類骨形成を示すアニリンブルーとPentachromeような組織学的な汚れを使用して、欠陥のスパンに沿った複数のセクションでは、ステンドグラスであるべき</li><li> Adobe Photoshopの、ではありませんすべてのエリア外に作物を使用して頭蓋冠欠損における</li><li>魔法の杖の機能を使用して、欠陥の領域でのde novo骨形成の画素数を決定し、コントロールまたは他の変数と比較します。 </li></ol> 6。代表的な結果脂肪組織は、臨床応用のための前駆細胞の生成に重要な役割を果たす可能性を秘めています。脂肪組織は、最小限の罹患率と死亡率を伴う比較的簡単な手順で収穫することができ、容易に利用可能な供給があるという点でユニークな利点を持っています。組織を採取し、収集されると、我々のプロトコルは、 図2に概説されている。脂肪由来の間質細胞は、洗濯の一連の手順、コラゲナーゼ消化し、遠心分離により単離されている。細胞が培養に播種されると、それらは、拡大して、in vitroで種々の分化プロトコルに入れ、またはin vivoで直接配置することができます。 C言語4ミリメートル臨界サイズの頭蓋冠欠損のreationは私たちの脂肪由来の間質細胞の骨分化能力を試験するin vivoモデルで簡単にアクセスして、再現性を提供します。 MicroCTを使用することにより、我々は、in vivoでの骨格組織の形成に従うと、私たちの介入の進捗状況（ 図3）を追跡することができます。重要なサイズの頭蓋冠の欠陥は、動物の生涯中に治らないし、我々は欠陥の約90％が8週間程度残る特許を参照してください。足場自体が（議論を参照）骨形成誘導特性を持っており、いくつかの骨の再生を誘導する能力を示してきた。細胞を含まない足場の配置の典型的な結果は、欠陥の約3分の1は、8週目でのde novo骨形成を持っていることを示しています。 variabはあるものの、脂肪由来の間質細胞の増強では、完全に欠陥の三分の二以上が約8週間で骨の再生が表示されます各動物と手術の間ility。骨格組織の形成は、組織学を介して定量化し、典型的な結果は、アニリンブルーとPentachrome染色（ 図3C）を介してASCを持つサンプルの増加類骨形成を示すことができます。加えて、我々は移植されたヒト細胞が頭蓋冠欠損におけるde novoの骨形成（ 図3D）の領域の近くに2週間でin vivoで染色を示すGFP標識hASCsの使用を介して基礎となるのde novo骨形成に寄与していることを示している。また、欠陥の面積（ 図3E）でヒト細胞の生存および貢献を示すために、ヒト特異的抗体を用いて免疫組織化学を使用しています。 <img alt="図1" src="/files/ftp_upload/4221/4221fig1.jpg" /> 図1 -脂肪吸引は、2つの層を持っています。最上層は、脂肪細胞とPRの大部分が含まれて底層はlipoaspiration手順の実行時に使用し生理食塩水が入っている間に細胞材料をocesses。 B - 基礎と硬膜を破壊することなく、右頭頂骨、4ミリメートルサイズの頭蓋冠の重要な欠陥のその後の創作を分離するために、頭蓋骨膜上に正中切開を介して頭蓋冠欠損の創造。 <img alt="図2" src="/files/ftp_upload/4221/4221fig2.jpg" /> 図2の進出、分化、in vitro および in vivo での使用に脂肪由来の間質細胞の単離から脂肪吸引の収穫とアプリケーションの概要。 <img alt="図3" src="/files/ftp_upload/4221/4221fig3.jpg" /> 図3 HYDを通してASCのアプリケーションとの臨界サイズの頭蓋冠欠陥の in vivoでの治癒に示す-MicroCTroxyapatitie足場（下段）。 ASCの群で有意に増加治癒を示すMicroCTから骨性治癒の定量 - コントロールは細胞（中段）Bなしの足場の配置と全く足場と欠陥（上段）と、欠陥を含んでいません。 C - アニリンブルーとPentachrome染色を通じて、ASCグループ（下の行）の増加した類骨形成を示す組織学。アニリンブルー、類骨汚れダークブルー用とPentachrome、黄色の骨汚れ用。 D - GFPで標識hASCSは、足場上に播種し、頭蓋冠欠損部に配置し、2週目に屠殺した。染色は欠陥の領域で再生に貢献ヒト細胞を示すため、GFP標識した抗体を用いて行った。 E - 2週間で欠陥の領域でヒト細胞の有病率を示すヒト核抗原の免疫組織化学では、 <a href="http://www.jove.com/files/ftp_upload/4221/4221fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-integrating+knockdown+and+customized+scaffold+design+enhances+human-adipose-derived+stem+cells+in+skeletal+repair.">Non-integrating knockdown and customized scaffold design enhances human-adipose-derived stem cells in skeletal repair.</a> Stem Cells. 29, 21028-21029 (2011).</li></ol>

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吸引脂肪から脂肪由来の間質細胞の単離2は 、これらの細胞は、細胞系統の多種多様に分化してきた。脂肪組織は中胚葉起源からであり、したがって、多能性脂肪由来間質細胞は、中胚葉系統に向かって可能性が高いアプリケーションで最も効果的であろう。骨格組織を生成する能力は、感染症、拒絶反応、および時間3以上の破壊を含む自家移植と合成材料の固有の限界のた...

<table border="1"><tbody><tr><td> 試薬の名称： </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td></td><td></td><td></td><td> 収穫を脂肪吸引 </td></tr><tr><td> PBS </td><td>ギブコ</td><td> 10010-023 </td><td></td></tr><tr><td>ハンクス液</td><td> Cellgro </td><td> 21から023-CV </td><td></td></tr><tr><td>コラゲナーゼ</td><td>シグマ</td><td> C6885-500mgの</td><td></td></tr><tr><td>セルストレーナーは100μm </td><td> BDファルコン</td><td> 352360 </td><td></td></tr><tr><td> 0.22μmのフィルター絆創膏トップ500ミリリットル</td><td>ミリポア</td><td> SCGPT05RE </td><td></td></tr><tr><td></td><td></td><td></td><td> 頭蓋冠の欠陥 </td></tr><tr><td> Z500ブラシレスMicromotorsUM50C </td><td>日本精工</td><td> NSKZ500 </td><td></td></tr><tr><td>円形のナイフ4.0ミリメートル</td><td> Xemax外科</td><td> CK40 </td><td></td></tr></tbody></table>

repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate

頭蓋顔面骨格の修復および再生には、幹細胞を用いた細胞ベースのアプローチによるde novoの組織形成の約束を提供しています。脂肪由来間質細胞（ASC）は、骨形成軟骨、脂肪細胞、筋原性と分化を受けることができる多能性幹細胞の豊富な供給源であることが証明された。多くの研究では、細胞送達のための様々な足場生体材料の使用により、生体内でのこれらの細胞の骨形成能を検討されている。それが実証されている骨伝導性ハイドロキシアパタイトでコーティングされたポリ（乳酸-co-グリコール酸）（HA-PLGA）のASCと足場シード、臨界サイズの頭蓋冠欠損、自発受けすることができない点で定義されている欠陥を利用することにより、動物の生涯にわたり癒し、効果的に堅牢な骨再生を表示することができます。 in vivoモデルこれは、骨組織を再生することを目的としたトランスレーショナルアプローチの根拠を示しています-携帯コンポーネントおよび生物学的マトリックス。このメソッドは、特定の組織の欠損の修復に向けた前駆細胞の究極的な臨床応用のためのモデルとして機能します。

Watch this Scientific Journal Video about Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate at JoVE.com

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

このプロトコルは、吸引脂肪や骨格再生を評価するための重要な4ミリメートルサイズの頭蓋冠欠損の作成から脂肪由来の間質細胞の単離を説明します。

吸引脂肪から採取した脂肪由来間質細胞を使用したクリティカル·サイズの頭蓋冠欠損モデルの修復

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

repair-critical-sized-calvarial-defect-model-using-adipose-derived

Research

JoVE Journal

Medicine

15.8K ビュー.  Stanford University. このプロトコルは、吸引脂肪や骨格再生を評価するための重要な4ミリメートルサイズの頭蓋冠欠損の作成から脂肪由来の間質細胞の単離を説明します。

ビデオ: Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

ヒト乳腺線維芽細胞からの癌関連線維芽細胞の実験的生成（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

医学

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

無限の地平線インパクターを用いた片側頚髄損傷の打撲のモデル

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

膜内骨化、仮骨延長術、またはCriticalサイズの欠損の治癒を評価するための剛安定骨折を作成する

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

血管内大動脈瘤の修復の新しいマウスモデル

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

瘢痕創傷修復のマウス胎児皮膚モデル

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

マウスの簡単な重要なサイズの大腿欠陥モデル

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

作成および糖尿病創傷治癒モデルにおける脂肪由来幹細胞 (ASC) シートの移植

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

根治的前立腺切除術後の勃起不全治療のための脂肪由来再生細胞の単離

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

壊死性腸炎の新生児BALB/cマウスモデル

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...