インテグラ皮膚再生テンプレートは、親切にインテグラライフサイエンス社によって提供された。作業を支援する資金源には：（。の人数15090007）この作品は、部分的にCIRM-BMBF早期トランスレーショナルII賞、ゲノム調節のためFONDAPセンターJTEとの両方によって賄われた。

足場の調製<ol><li> 12ミリメートル生検パンチを使用して、足場のサンプルを生成します。 </li><li>足場内に生物活性分子または細胞を導入するために、穏やかに滅菌ガーゼでそれらを絞ることによって足場をドレイン。次いで、目的の生物活性分子または細胞を含む溶液の160μLを加えることによって足場を再水和する。そのようなMTTアッセイなどの代謝アッセイを通して細胞と生物活性化の成功をダブルチェックしてください。 </li><li>必要に応じて、ステップ1.2で説明したように、再び再水和を介して、フィブリン - トロンビン溶液またはヒドロゲル中に、例えば、それらを提供することにより、足場の内部でテストすべき化合物または細胞を固定。 注記：化合物または細胞が機械的に材料に付着していない場合、このステップでは、便利であることができる。一つは、また、化合物の放出動態を制御することができます.. </li><li>カット時によって、全ての実験および対照群ではそれぞれの足場の下に置かれるチタン化メッシュを、準備しますgのうち円形の小片、直径約14ミリメートル。 注：チタン化メッシュが創傷床から再生された組織を区切るために必要な物理的境界として機能します。 </li></ol> 2。動物<ol><li>提示されたモデルの実装に先立ち、対応する動物保護法を参照し、地元当局からの許可を得る。本研究では、すべての実験は、現在のドイツの動物福祉の行為に応じて、マウスを用いて行ったし、オーバーバイエルン（RegierungフォンOberbayern）特別区政府によって承認されました。 </li><li>動物や歪みを慎重に選択します。モデルはマウスで確立されたが、それは、ラット、ウサギまたはブタのような他の一般的な動物に変換することができる。 </li><li>あなたは毛皮動物で作業している場合には、足場の移植前の動物の背中を剃る。 注：この作品は20グラムと25グラム、しかし異なる年齢とbの間の体重の生後6〜8週のマウスを使用していますODY重みを使用することもできる。 </li></ol> 3麻酔<ol><li>無菌条件下で動作を行う。動物の正常な体温を維持するために、加温マットを使用する。 </li><li>以前は吸入麻酔動物は0.05〜0.1ミリグラム/ kg体重、皮下8時間毎の用量でBuprenorphinを受ける。 </li><li>吸入麻酔（イソフルラン）の下で動物を置きまたは同等の標準的な手順を適用します。優しく動物のつま先を絞ることによって、または皮膚ピンチのいずれかを介して麻酔を確認してください。 </li><li> exsiccosisを防止するために、0.5ミリリットルの動作の開始時に生理的食塩水を皮下注射する。 </li></ol>スキンの4切除<ol><li>腹臥位で動物を置き、先端の細いパーマネントペン（ 図1A）、マウスの背中の正中線をマーク。 </li><li>切除範囲を定義します。 ふぃぎゅに示す領域内の欠陥を置きます1Cを再び。欠陥があまりにも尾側に配置された場合、動物はドレッシングや足場を除去する傾向であることに注意してください。 </li><li>の10mm生検パンチ（ 図1B）を使用してバイラテラル欠陥の周りを作成する。パンチを慎重に皮膚に押し付けされるべきであり、皮膚を通って完全に切断するのに使用してはなりませんが、切除領域（ 図1C＆1E）を描写する。 </li><li>静かにピンセットでマークされた肌を持ち上げ、微細手術用ハサミ（ 図1F）を使用してマークされた円に沿って切開する。出血の場合には、慎重に、滅菌ガーゼで圧迫する。切除のステップバイステップの説明は、 図1に示されている。 注：欠陥が肌の弾力性に起因する欠陥の拡大を補償するために、足場を生成するための1より小さくパンチを使用して作成されます。 </li></ol> 5。足場移植<ol><li>ティタのためのスペースを作るためにデバイスをメッシュ2-4ミリ（ 図2A）のための創傷境界で、皮膚とその下の組織の分離を拡張します。 </li><li>創傷床の上に直接欠損部にメッシュをチタン化し、傷のエッジ（ 図2AおよびB）の下に置きます。 </li><li>メッシュの上に直接足場を置きます。 </li><li>足場（ 図2C）にわたって少しの縁を残して、4〜6つの結び目を、隣接する巻回エッジに足場を縫合する。 注：生分解性縫合糸の使用は避けてください。 </li><li>創傷領域（ 図2D）の監視を可能にしながら足場を保護するために、欠陥の上にドレッシング透明傷口を縫合。 </li></ol> 6。術後ケア<ol><li>ドレッシングや足場の相互操作を回避するために、ケージごとに1つのマウスを保管してください。 </li><li>運動活動、体重、苦痛の徴候、許容範囲を観察することによって、日常的に動物の一般的な状態を評価するドレッシングと自動傷に。また、出血、感染症の局所および全身兆候およびドレッシングの位置のための創傷領域を監視します。 </li><li>動物の苦痛を最小限にするために、0.05〜0.1ミリグラム/ kg体重または同等の鎮痛薬の用量で毎日Buprenorphinを注入する。 </li><li>マウスは創傷被覆材を除去または損害賠償場合は、麻酔下でそれを交換するか、再接続します。 </li></ol>足場の7。安楽死や植<ol><li>希望の時点で、ペントバルビタール（150mgの/ kg）を過剰投与により動物を生け贄に捧げる。 </li><li>足場および可能性（ 図3A）、バック、マウスの皮膚の多くを含むべきである油性マジック、と皮膚切除のサイトをマークします。 </li><li>ハサミやメスでマークされた線に沿って皮膚を切開。下にある組織から鈍準備（FIを通じて、足場およびチタン化メッシュを含む全体の肌を、デタッチグレ3B）。 </li><li>ペトリ皿に伸ばし移植組織を置きます（ 図3CおよびD） </li></ol>血管網の8。可視化と定量化<ol><li>徹照： <ol><li>白色光（100ワット、標準の電球）の強力なソースの上にシャーレを搭載することにより、透視デバイスをセットアップします。 </li><li>デジタル画像を得るために、イルミネーターの上にデジタルカメラを固定します。 </li><li>逆さまに透視デバイス上のペトリ皿上のサンプルを置きます。 </li><li>マクロモードでの完全な足場の写真を撮るだけでなく、正常な皮膚の等しいサイズの領域。さらにデジタル解析用のTIFF形式で画像を保存してください。 </li><li>さらに生化学的または組織学的分析のために組織を保存します。 </li></ol></li><li>デジタルセグメンテーションおよび定量： <ol><li> FRを求めることができるVesSeg-Toolソフトウェアを、ダウンロードしてインストールします：における電荷のee <a href="http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255">http://www.isip.uni-luebeck.de/index.php?id=150&amp;L=2%255</a> 。 </li><li> VesSeg-Toolソフトウェアで画像を開きます。 </li><li>デジタル解析（画像→選択）のための足場で覆われた画像の領域を選択します。 </li><li>オプション「ヒステリシスしきい値」（計算血管強化→トップハット変換→画像→血管強調フィルタ→ヒステリシス閾値処理）を使用して血管のコントラストを増やします。 </li><li>血管マップ（画像→血管強調フィルタ→ヒステリシス閾値化→しきい値の境界線）のセグメント化を進めます。でも、リモートで血管様であるすべてのピクセルなので、第一の閾値（「船舶保険」）を選択して、容器とラベルされます。第二の閾値（「背景カバレッジを」）を選択したので、特に血管様であるピクセルのみが容器としてラベル付けされます。 </li><li>非捕捉血管を追加するために、通常はそのような毛や縫合糸など細長い、血管様構造体である一般的な偽陽性構造を排除するために手動で自動分割案を確認してください。 </li><li>血管の長さと血管（バイナリ画像統計→画像→画像の統計量）でカバー全体の面積を計算します。 注：血管の長さを計算するために、結果として得られる血管マップ内の血管が1画素20の幅でラインに間引く。 </li></ol></li><li>天然組織に100％の値を割り当てる、足場と同じパラメータの下でネイティブ皮膚領域を分析し、その値に足場を関連付ける。白画素の割合が、正常組織では60％と足場30％である場合、例えば、それは足場の50％の血管新生を表す。注：白い船カバレッジが足場の周りの正方形の面積に割り当てられている。白画素カバレッジconsiの数を調整円の面積は、正方形（4×R 2）よりも有意に小さい（πさ×R 2）であることをダリング。 </li></ol>

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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=Use+of+human+mesenchymal+cells+to+improve+vascularization+in+a+mouse+model+for+scaffold-based+dermal+regeneration.">Use of human mesenchymal cells to improve vascularization in a mouse model for scaffold-based dermal regeneration.</a> Tissue Eng Part A. 15 (5), 1191-1200 (2009).</li><li>Condurache, A., Aach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+segmentation+in+angiograms+using+hysteresis+thresholding.">Vessel segmentation in angiograms using hysteresis thresholding.</a> Proceedings of the Ninth IAPR Conference on Machine Vision Applications. , 269-272 (2005).</li><li>Ega&#241;a, J. 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=Ex+vivo+method+to+visualize+and+quantify+vascular+networks+in+native+and+tissue+engineered+skin.">Ex vivo method to visualize and quantify vascular networks in native and tissue engineered skin.</a> Langenbecks Archives of Surgery. 394, 349-356 (2009).</li></ol>

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生体材料内の血管新生の過程を研究するための新しい信頼性の高い方法の開発が求められて組織工学構築物中の血液灌流を改善するのに成功したアプローチを確立する必要がある。 ex vivoで可視の足場血管新生を製造するための一般的な方法は、高解像度のツールを提供する顕微鏡の使用を含む。ほとんどの場合、しかし、この方法は、小さな組織領域の分析に制限され、高価で時間?...

ここ数十年では、組織工学は、体自身の細胞1を組織欠損に代わる新たな治療オプションを開いた。組織再生の生理学的プロセスをサポートするために、足場を創傷床からの細胞が成長し、欠陥2,3を復元することができるシナリオを提供し、生分解性構造として設計されている。 不十分な血管新生は、バイオ人工足場4の臨床的ブレークスルーをバック保持している主な障害であると考えられている。細胞の内方成長で、栄養と酸素が増加し、材料の血管新生の需要が不可欠となる。不十分な血管新生または遅延は、したがって、組織工学製品5の中心壊死につながることができます。また、血管が免疫コンピテント細胞を提供し、再生領域における代謝残留物を除去する。高い感染率と低い再生は唯一である足場6,7の血管新生を増加させることによって回避することを目的としている組織工学において観察不十分な血液灌流の結果の一部。 生体材料自体と足場の微細構造の重要な役割に血管新生のフォーカスを改善することを目指していくつかの戦略。 8,9を復元することにより（再）、 再生に修理から治癒過程をシフトに最も近い生理学的特性を有する組織を生成する際に新たなアプローチを開発するための集中的な研究努力がある。その再生能力に関しては研究し、評価した生体材料は、コラーゲン、フィブリン、キトサンおよびアルギン酸10,11が含まれていた。これらの生体材料は、組織の脱細胞化、自己集合、ラピッドプロトタイピングやエレクトロ12のような異なる戦略を用いて新たな足場を構築するための骨格として使用され、組み合わせることができる。 ENHするためにボディの自身の再生能力をANCE、足場は、生物活性化することができます。組換え血管新生増殖の組み込みは、足場の血管新生を改善することが示されている14のような因子のために13または遺伝子をコードするベクター因子。幹細胞の使用は、広く間葉ストローマ細胞および内皮前駆細胞が、最も注目を集めている15,16血管新生を改善するための有望な戦略であることが示されている。他のアプローチは、移植前17にプレハブ血管網が含まれている構造物を構築しようとします。足場の設計とその生物活性化に集中的な取り組みにもかかわらず、戦略は、臨床的に有意なレベルでの血管新生を改善していないと、大規模な火傷での皮膚代替物を除いて、臨床ルーチンへの生体工学材料の翻訳はためらいがちに18を行われている。 理由の一つなぜ血管新生人工組織構造物を依然として未解決の問題であり、in vivoでのアプローチにおける新技術の成功を評価することが困難である。 インビトロ実験は、足場の血管新生の可能性の重要な洞察を提供し、適切な動物モデルは、このような材料の生体適合性などの重要なパラメータを研究するために必要とされる、治療の安全性および有効性と、特に重要な、組織の血管新生ができるが構築する。したがって、in vivoでの血管ネットワークを視覚化し、定量するための信頼性の高いツールが不可欠である。 本研究では、外植足場内部の血管網の可視化および定量化を可能にする、シンプルで信頼性の高い方法を提示する。この方法は、組織の透視やデジタルセグメンテーションに基づいています。この方法は非侵襲的であるため、ターゲット材料のさらなる分子および組織学的分析を可能にする。

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			<td height="40" style="height:40px;width:319px;">Ethilon P-3 13 mm 3/8 circle 5-0</td>
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			<td style="width:119px;">698G</td>
			<td>Ethilon polyamid-6 precision point-reverse cutting suture</td>
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			<td height="40" style="height:40px;width:319px;">Biopsy punches (10 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1050</td>
			<td></td>
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			<td height="40" style="height:40px;width:319px;">Biopsy punches (12 mm)</td>
			<td style="width:223px;">Xiomedics, Acuderm inc., Fort Lauderdale, FL, USA</td>
			<td style="width:119px;">P1250</td>
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			<td height="40" style="height:40px;">Digital camera&#160;</td>
			<td style="width:223px;">Ricoh, Hannover, Germany</td>
			<td style="width:119px;">Cx1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Gazin Mullkompresse&#160;</td>
			<td style="width:223px;">Lohmann und Rauscher, Neuwied, Germany</td>
			<td style="width:119px;">13622</td>
			<td>Sterile gauze (10 cm x 10 cm)</td>
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			<td height="40" style="height:40px;">Double-layer collagen-based scaffold (8 &#39;x 10&#39;)</td>
			<td style="width:223px;">Integra Life Science Corporation, Plainsboro, NJ, USA</td>
			<td style="width:119px;">88101</td>
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			<td height="40" style="height:40px;">Isoflurane, liquid-gas for inhalative anesthesia&#160;</td>
			<td style="width:223px;">Baxter, Unterschleissheim, Germany</td>
			<td style="width:119px;">100196040</td>
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			<td height="40" style="height:40px;">Pentobarbital, 16 g / 100 ml</td>
			<td style="width:223px;">Fa. Merial, Hallbergmoos</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="40" style="height:40px;width:319px;">Nuri Nu/Nu Nude mice, CrLNU-Foxn1nu</td>
			<td style="width:223px;">Charles River, Sulzfeld, Germany</td>
			<td style="width:119px;">Strain code 088</td>
			<td>Athymic nude mice, 6 to 8 weeks of age and with a body weight between 20 to 25 g&#160;</td>
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			<td height="40" style="height:40px;width:319px;">Buprenorphine (0.3 mg/ml)</td>
			<td style="width:223px;">Essex Pharma GmbH, Munich, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Titanized mesh (15 cm x 15 cm), extralight</td>
			<td style="width:223px;">PFM Medical AG, K&#246;ln, Germany</td>
			<td style="width:119px;">6000029</td>
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			<td height="40" style="height:40px;width:319px;">Tissucol Duo S Immuno 2 ml</td>
			<td style="width:223px;">Baxter Germany GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;">B1332020110614</td>
			<td>Fibrin-thrombin solution&#160;</td>
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			<td height="40" style="height:40px;">Transparent adhesove drape (30.5 cm x 26 cm)</td>
			<td style="width:223px;">KCI Medical Products, Wimborne Dorset, UK</td>
			<td style="width:119px;">M6275009/10</td>
			<td></td>
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a full skin defect model to evaluate vascularization of biomaterials in vivo

不十分な血管新生は、組織工学構築物の臨床的成功を制限する主な要因の一つであると考えられている。血管新生を改善することを目指す新たな戦略を評価するために、信頼できる方法は、可視バイオ人工足場への新しい血管の内殖を行い、結果を定量する必要がある。過去数年にわたり、私たちのグループは、透視によって血管の直接可視化を可能にし、デジタルセグメンテーションを通じて定量化する可能性を提供する、完全な皮膚欠損モデルを導入しています。このモデルでは、人は外科的にマウスの背中に完全な皮膚欠損を作成し、テスト材料に置き換えます。目的の分子または細胞は、それらの潜在的効果を研究するために、そのような材料に組み込むことができる。自分の選択した観測時間の後、材料は、評価のために外植される。両側性の創傷は、目の内部の比較を行う可能性を提供するでの学習に必要な動物の数を減少させるだけでなく、個人間で成果物を最小限に抑えます。他のアプローチと比較して、私たちの方法は、簡単で信頼性が高く、費用対効果の分析を提供しています。私たちは、別の生体材料、バイオ活性化のアプローチの血管新生をテストする際に、高解像度のスクリーニングを行うためのルーチンツールとしてこのモデルを実装している。

信頼性のある国間の完全な皮膚欠陥、皮膚は、研究中の生体材料（ 図2）で置き換えることができるマウス（ 図1）で作成することができる。ここでは、大きな合併症は、感染や異物反応の巨視的兆候もないが、手術手順の間または後に観察されない。マウスはそれを削除する際にまれに、足場は失われます。創傷収縮は、（ 図3）が観察されなかった。?...

Watch this Scientific Journal Video about A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo at JoVE.com

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

血管新生は、正常組織工学におけるアプローチの鍵となります。したがって、信頼性の高い技術は、組織構築物における血管網の発達を評価するために必要とされる。ここでは、可視化し、 生体内で血管新生を定量化するためのシンプルで費用対効果の高い方法を提示する。

生体材料の血管新生を評価するために、完全な皮膚欠損モデル<em&gt;インビボ</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

a-full-skin-defect-model-to-evaluate-vascularization-biomaterials

Research

JoVE Journal

Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

12.1K ビュー.  Technische Universität München. 血管新生は、正常組織工学におけるアプローチの鍵となります。したがって、信頼性の高い技術は、組織構築物における血管網の発達を評価するために必要とされる。ここでは、可視化し、 生体内で血管新生を定量化するためのシンプルで費用対効果の高い方法を提示する。 

ビデオ: A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三次元ヒト皮膚は、モデルを再構築：ノーマルスキンとメラノーマ進行を勉強するツールを

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

生物工学

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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

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

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

三次元の全層皮膚等価物と自動傷つけ処理の発生

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

Sandwich-like Microenvironments to Harness Cell/Material Interactions

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

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

サンドイッチ状の微小環境は、Cell /材料の相互作用を活用します

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

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

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

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

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

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

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

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

振動触覚刺激に随意運動反応の適時性と精度の評価方法

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

ゼブラフィッシュ胚モデルにおける生体内可視化と生体材料の生体解析黄色ブドウ球菌感染症

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

ラットのカスタム創外固定器で安定した信頼性と再現性のある重要なサイズ分節大腿骨欠損モデル

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

皮膚における遺伝的にタグ付けされたレポーター細胞の生体内2色2光子イメージング

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

骨置換材料における骨成長と新生血管の評価のためのウサギにおける骨増強の卵子モデル

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...