この作品は、（HPL）は、NIH助成金R01 GM087609、（MTL）は（HPL）のアンソニー·シュウ、NIHのグラントU01のHL099776に敬意を表しイングリッド·レイとビル·シュウからの贈り物、Hagey研究のための助成金によって部分的にサポートされていました小児再生医療と（MTLとHPLへ）オーク財団。 GGWはスタンフォード大学医学部、スタンフォード医学者研修プログラム、及びNIGMS訓練助成金GM07365によってサポートされていました。 MSHはCIRM臨床フェロー研修グラントTG2-01159によってサポートされていました。 WXHはサーノフ循環器財団からの資金によってサポートされていました。

注：この論文で説明されているすべての手順は、実験動物管理上のスタンフォードの紛争処理パネル（APLAC）によって確立されたガイドラインに従って実施されている。 1.時限妊娠 - 過排卵技術（図1） 注：正確にE16.5とE18.5での胎児手術のためのマウス胚の妊娠期間のタイミングは非常に重要である。このセクションの我々の詳細には過剰排卵を誘発するために妊娠馬血清（PMS）およびヒト絨毛性ゴナドトロピン（HCG）注射を用いて、マウスの妊娠のタイミングのために私たちのプロトコル。 <ol><li> 1日目のために一時と午後3時間100μlのPBSの容量でPMSの3.0から5.0国際単位（IU）でメスのマウス（&lt;ケージ当たり5）を腹腔内（IP）を注入します。 </li><li> 12：00 3日目（四〇から五PMSの注射後時間〜47）の2:00 PMの間に、100μlのPBSの容量でHCGの3.0から5.0 IUとメスのマウスIPを注入する。 注：HCG注射を誘導<html>排卵の約12時間後に注射。 </li><li>直後にHCGの注射後、8-16週齢の雄と雌交尾。 注：我々は、典型的には、個々の男性のケージに2人の女性を置く。 </li><li>胎児の年齢E0.5として - （10:00 AM 6時00分）とレコード4日目の朝に、男性とは別の雌。 注：ワギナプラグは、この時点で確認することができます。しかし、膣のプラグの観察は、妊娠を保証するものではありませんし、何プラグが観察されない時に女性が妊娠することができます。妊娠が妊娠年齢のE16.5及びE18.5での目視検査および/または触診によって一般的に観察可能であることを考えると、4日目の朝に膣栓をチェックすると、必ずしも必要ではありません。我々の経験では、歪みに応じて、超排卵の雌の約30から50%はここに記載されている技術を使用して妊娠。 </li></ol>pgの &quot;/&gt; 過排卵技術については、図1の回路図。マウスでの過剰排卵時限妊娠の回路図を示す手順。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> E16.5およびE18.5胚2.マウス胎児手術（背傷つけ処理）（図2） <ol><li>手続きの前に、70％のイソプロピルアルコールで手術室や機器の全ての表面をきれいに。また、それらをオートクレーブ処理により手順で使用されるすべての手術用品や楽器を殺菌。いくつかの機関は、ホットビーズ滅菌のその後の使用を可能にすることができる。操作ごとに、ガーゼや手術器具を含む無菌パックを使用しています。 </li><li>毎分1リットルでのメンテナンス麻酔に続いて、毎分2リットルで2.5％イソフルラン/酸素混合物の下で妊娠中の母親（胎児の年齢E16.5またはE18.5）で麻酔を誘導する。 </li><li> CONへしっかりと適切な麻酔、マウスの深いペダル反射が抑制されていることを確認し、腹臥位でマウスを置く。 </li><li>手術中眼刺激や乾燥を防ぐためにそのようなPuralubeとして獣医眼軟膏を適用します。 </li><li> 30秒（ 図2A）より、もはや用脱毛クリームの光アプリケーションを投与することにより、腹部を準備します。 </li><li>ポビドンヨードとアルコール（ 図2Bおよび2C）との無菌手術のために腹部を準備します。 </li><li>顕微はさみ（ 図2Dおよび2E）を用いて顕微鏡下で正中開腹を行います。 </li><li>静かに手術（ 図2F）用に選択子宮や胎児を公開します。 </li><li>鈍い先端の針を用いて温かい（38℃）リン酸緩衝生理食塩水（PBS）で手術野を洗浄 注：One慎重大口径の針の先端を折り曲げて作製することができる。 （ 図2Gおよび2H）。 </li><li>許可するようにした胎児を置き背にフルアクセスだ。 </li><li>意図された背側創傷（ 図2I）のサイトの上にある子宮を通して7-0ナイロン縫合糸を使用した巾着ステッチを渡します。脊髄の左または右へ、および大血管を欠く子宮壁の領域における背の領域にわたって位置巾着。 </li><li>巾着（ 図2J）の中央に子宮壁と羊膜嚢を通じて3mmの切開を行います。 </li><li>温かい（38℃）PBSで切開部位を洗浄する。 </li><li>顕微はさみを使用して、胎児の背に、単一の全層切除傷、長さ約1ミリメートルをカット。 </li><li>そっとブロット切開部位は、綿チップアプリケーターで乾燥させます。 </li><li>傷（ 図2K）の場所をマークするために、創傷部位に3μlのボリューム墨汁の皮下に注入します。 </li><li>インクが創傷部位内に保持されていることを確認するために温かい（38℃）PBSで灌漑。 </li><li>外科助手の私を持っているnject巾着など羊膜嚢に鈍い先端10のG注射器を通して温かい（38℃）、PBS（ 図2L）を閉じている。巾着の閉鎖が完了します（ 図2M）を近づくと注射器を撤回。 </li><li>静かに腹腔（ 図2N）に子宮を返す。 </li><li>エバートの皮膚や腹膜。 </li><li>温かい（38℃）PBSで腹腔を外科助手を灌漑している。 </li><li>皮膚や腹膜ステープルすばやく閉じる腹部が（ 図2O）閉じた。標準的な閉鎖は二層で行われる。腹膜および第2層における一方の層、皮下組織および皮膚の腹筋。即時の犠牲と胎児の収穫のために、私たちのデモが1層で閉鎖を示している。 </li><li> 30分間または動物が胸骨横臥位を維持するのに十分な意識を取り戻すまで、37℃に設定した暖かいインキュベーター内で観察下に動物を置きます。 </li><li> retにしないでくださいそれは完全にプロシージャから回復するまで、他の動物の会社に動物をURN。 </li><li>麻酔からとその後の48時間の間に覚醒時には、ブプレノルフィンの皮下注射（0.05 mg / kgを）疼痛評価に基づいて、必要に応じて鎮痛のために12時間ごとに管理する。必要に応じて追加の術後疼痛緩和のために皮下注射を介してカルプロフェン（5ミリグラム/ kg）を管理します。 </li><li>ケージに動物を返し、食物と水を自由にそれらを提供する。 </li><li>痛みの症状のために密接に監視します。 </li><li> 48時間後に手術、イソフルランと収穫負傷胎児の過剰摂取で妊娠中の母親を生け贄に捧げる。これを行うためには、5％以上にイソフルラン濃度を調整し、呼吸停止後に1分間曝露を維持する。頸椎脱臼で安楽死を確認してください。年齢をマッチさせたコントロールのための無傷胚を収穫。後期胚は、次のような動物実験委員会の勧告、と一貫安楽死の別のメソッドを持っている必要がありますデ人頭、頸椎脱臼、または化学薬品注入。 </li></ol><img alt="図2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> 図2.回路図マウス胎児外科。E16.5及びE18.5マウス胚で負傷背部するための一般的な手順。（A）マウスの腹部の脱毛。（BとC）マウスの腹部の調製。（D）顕微鏡外科に使用子宮を通じて巾着縫合の手順。（E）ミッドライン開腹。（F）子宮の露出を。鈍先端針の（G）の作成 ​​。温かい生理食塩水で子宮の（H）灌漑。（I）の作成​​。（J）切開壁と1ミリメートルフル層切除創傷世代。（K）墨汁の皮下注射。（LとM）巾着縫合の閉鎖。（N及びO）腹部の閉鎖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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The authors have nothing to disclose.

ここで紹介するの外科的プロトコルは、最初に私たちの研究室10が2006年に出版さ胎児マウスの瘢痕治癒の切除モデルについて説明します。切除創傷11の他の確立されたモデルに加えて、胎児マウス瘢痕治癒の切開モデルが同様に存在する12,13。サル、子羊、ウサギ、オポッサム、ラットで胎児の瘢痕創傷治癒の調査は、14〜17を報告されている。しかし、マウスは、それらの比較的低いあたりの日当ケージコストと十分に特徴付けられたゲノムに胎児の瘢痕創傷治癒を探索するのに理想的なモデルを表す。また、関数の時間的および空間的な遺伝的損失/利得を正確瘢痕修復のメカニズムを解読するための機会を提供するトランスジェニックマウス系を用いて、胚発生の間に達成することができる。 これらの利点にもかかわらず、マウスは、それらのサイズが小さい胎児手術中に特定の技術的な課題を提示する。与えられた妊娠中の母親に麻酔のストレス、細心の注意を投与されているイソフルラン/酸素レベルを監視するために注意が必要です。毎分1リットルのメンテナンス麻酔に続いて、毎分2リットルで2.5％イソフルラン/酸素の混合物を慎重に接着されるべきである。この重要な要因の偏差または変動はかなり罹患率に影響を与えることができます。巾着縫合糸の適切な配置は、手術の最も技術的に困難な側面を表す。正しくこのステッチを配置しないと羊水とPBSポスト決算の漏れが発生します。子宮内の流体の不十分な体積は、胚への外傷になり、早期子宮収縮を誘発し得る。このため、子宮口時に失わ羊水の交換も、この外科モデルにおける重要なステップである。ケアは、巾着が閉じているように継続的に暖かいPBSを排出するように注意しなければならない。これらの要因は重要なトラブルシューティングが必要であり、我々はこれらの詳細にその注意を強調する成功した手術の可能性が高くなります。しかし、それらの経験豊富な外科の手では、E16.5胎児で約40％の罹患率のために計画する必要があります。 胎児の瘢痕創傷治癒を理解することは、開発の成人の段階で線維形成挙動のin vivoでの変調を目的としたトランスレーショナ医学の値を保持します。大人の皮膚と比較して、胎児の皮膚は、現像真皮減少引張強度、発生期の毛包、および異なるECM成分2,4を有している。胎児の皮膚は、大人の皮膚4,9と比較して成人の皮膚、より少ない少ない成熟肥満細胞、ケラチン8および19、およびヒアルロン酸の高いレベルの発現と比較してIを入力するためのコラーゲンタイプIIIの上昇比を有する。胎児における瘢痕治癒のメカニズムを解読することにより、我々は、回生表現型への成人における線維症の原因と分子·細胞経路を操縦し始めることができます。羽化OマウスでトレースF系統と他の遺伝的ツールは、胎児の瘢痕創傷治癒の調査のための有望な新たな道を開いた。罹患率の本質的に高い率と胎児手術に関連した早期子宮収縮を考えると、胎児の瘢痕皮膚の調査は正確で再現性の外科的モデルを必要とし、in vivoで創傷治癒。ここでは詳細E16.5（瘢痕）とE18.5（瘢痕化）マウス胚の背で胎児の瘢痕創傷治癒の再現可能なモデル。

胎児皮膚創傷は、妊娠後期1まで急速かつscarlessly癒す。胎児の瘢痕創傷修復は、正常な組織構造および機能の再生によって特徴付けられる。瘢痕化の表現型への瘢痕からの移行は、ヒトにおけるマウス2,3における胎生18日（E18）の周りに妊娠後期に起こる。成人と比較して、胎児の創傷修復を迅速上皮、結合組織の堆積、および線維芽細胞の遊走によって特徴付けられる。 多くの研究は、初期の胎児の発達中の瘢痕創傷治癒の現象の可能な説明を提供してきました。炎症は、大人の創傷修復の基本的なコンポーネントです。しかし、胎児の創傷は、急性炎症4の欠如によって特徴づけられる。これは胎児の段階で免疫系の機能的な未熟さの結果であるかどうかは不明なままである。最近の研究では、豊富に違い、マットと示唆urity、およびE18胎児の皮膚対E15における肥満細胞の機能は、少なくともマウス3において、瘢痕の表現型への移行に関与し得る。他の研究では、プロパティと胎児および成人の創傷マクロファージの存在量の違いが胎児の創傷修復5中に、通常の細胞外マトリックス（ECM）の改革のために責任があることを断定。 胎児および成人の発達中の環境要因の違いはまた、創傷の修復に影響する場合があります。 Longakerらは、胎児から創傷液が成人の創傷液6でなしに比べ、ヒアルロン酸刺激活性の高いレベルを持っていることを示した。従って、胎児の創傷環境におけるヒアルロン酸の高いレベル、細胞運動性および増殖につながる微小環境を促進するグリコサミノグリカンは、初期の胎児の発育中に見られる瘢痕の表現型の原因である可能性がある。フェタチーズはその事実に証拠ポイントのその他のラインLの創傷環境は、比較的低酸素および成長因子7に富む無菌羊水中に浸漬される。しかし、決定的な答えは、線維性の修復に瘢痕再生への移行をトリガー胚形成の間の重要なイベントや要因のために提供されていない。 胎児に瘢痕治癒の責任のメカニズムを理解することは、正確で再現性のあるモデルを必要とする。ここでは詳細E16.5（瘢痕）とE18.5（瘢痕化）マウス胚の背で胎児の瘢痕創傷治癒の再現可能なモデル。さらに、このモデルの小さな変化は、胎児の創傷及び皮膚8,9の遺伝子発現解析など、さらなる研究の数を実行するために利用することができる。正確なタイミング妊娠がこの胎児の瘢痕創傷治癒モデルの成功した要約のために重要であることを考えると、私たちは過剰排卵時限妊娠についても詳細私たちのプロトコル。

<table><tbody><tr><td>7-O MONOSOF Suture</td><td>eSuture</td><td>SN-1647G</td><td></td></tr><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Autoclip 9 mm</td><td>Texas Scientific Instruments</td><td>205060</td><td></td></tr><tr><td>Insulin Syringe</td><td>Thermo Fisher Scientific</td><td>22-272-382</td><td></td></tr><tr><td>Black Pigment</td><td>AIMS</td><td>242</td><td></td></tr><tr><td>BD Safety-Lok 3 ml Syringe</td><td>BD Biosciences</td><td>309596</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Life Technologies</td><td>10010-049</td><td></td></tr><tr><td>OPMI-MD Surgical Microscope</td><td>Carl Zeiss Surgical Inc</td><td></td><td></td></tr><tr><td>Pregnant Mares Serum (PMS)</td><td>Millipore</td><td>367222</td><td></td></tr><tr><td>Human Chorionic Gonadotropin (HCG)</td><td>Sigma-Aldrich</td><td>CG10</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr></tbody></table>

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.

組織学的分析のために、背側の皮膚E16.5及びE18.5における皮膚創傷マウス胚を4％PFAで固定し48時間後創傷、収穫されるべきであり、パラフィン包埋した。蛍光トランスジェニックモデルでは、10月との凍結保存が適切であり得る。細胞および結合組織の構造を可視化するために使用できるいくつかの汚れがある。ヘマトキシリンとエオシン、核青と好酸球の構造（ すなわち 、細胞質と細胞外コラーゲン）を染色する二色染色である、赤、ピンク、オレンジのさまざまな色合い。マロリー·トリクロームは、結合組織の周囲の細胞を区別するための最適な、アニリンブルー、酸性フクシンオレンジ色のGからなる三色染色である。 <img alt="図3" src="/files/ftp_upload/52297/52297fig3highres.jpg" /> 図3.瘢痕E16.5胎児創傷の組織学。 （A）ヘマ ​​トキシリンおよびエオシン染色の再veals完全な再上皮化および炎症細胞の数の穏やかな増加が存在する（矢印）。バー= 25;（100倍;バー= 100μm）は（B）エオシン染色は、再生毛包（矢印）（400倍の周りに墨汁（矢印）を示している。バー=25μmの）;μm）は（C）マロリーのトリクローム染色は、毛包（400倍の存在と細かい網状真皮コラーゲンパターンを明らかにする。コルウェルら 10から許可を得て転載<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig3large.jpg" target="_blank">。この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 背側切除創傷は、適切なサイズ（1ミリメートル）と深さ（全層）である場合は、ヘマトキシリン​​及びエオシン染色は、最小限の瘢痕化、完全な再上皮化、および炎症の数のわずかな増加とそのE16.5の皮膚治癒を明らかにする細胞（ 図3A）。さらに、これらはwounDSは約正常な皮膚のアーキテクチャで癒し、（矢印; 図3B）損傷の部位の中の毛包を再生させる含まれている必要があります。新規に作成した創傷への墨汁の適切な適用は、損傷部位（; 図3B矢頭）でのインクの堆積をもたらすべきである。最後に、トリクローム染色は、非瘢痕真皮（ 図3C）の細かい網状真皮コラーゲンパターン特性を明らかにする必要があります。 <img alt="図4" src="/files/ftp_upload/52297/52297fig4highres.jpg" /> 図4.瘢痕E18.5胎児創傷の組織学。 （A）ヘマ ​​トキシリンおよびエオシン染色は損傷部位で真皮におけるエオシン染色の増加（矢印）を明らかにする。（200X;バー= 50μm）を（B）マロリーのトリクローム染色は、緻密皮膚コラーゲン（矢印）（400倍を示しています。バー= 25ミクロン）。コルウェルらから許可を得て転載。10 E18.5で行われた背側の傷のE16.5、ヘマトキシリンおよびエオシン染色で行われた傷と比較して、48時間後に創傷が損傷部位（ 図4A）での正常な皮膚構造の喪失と密な傷跡を明らかにする必要があります収穫。同様に、トリクローム染色は、無秩序なコラーゲン沈着（矢印; 図4B）の密なパターンを明らかにしている。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52297/52297fig4large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

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-mouse-fetal-skin-model-of-scarless-wound-repair

Nanyang Technological University

Research

JoVE Journal

Medicine

12.2K ビュー.  Stanford University School of Medicine. 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.

ビデオ: A Mouse Fetal Skin Model of Scarless Wound Repair

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

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

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

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

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

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 ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

創傷治癒のマウスモデル

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

医学

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 ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

後部皮下ポリビニルアルコールスポンジの移植と切除テール皮膚創傷モデルを用いた急性創傷治癒の評価

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

免疫・感染学

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

メカノトランスダクション駆動型ヒト様肥厚性瘢痕のマウスモデル

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

マウスモデルにおける創傷閉鎖速度を評価するためのデジタルプラニメトリー

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

同種異系皮膚移植片で再建された火傷のマウスモデル

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

生物学

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

瘢痕創傷修復のマウス胎児皮膚モデル

要約

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同種異系皮膚移植片で再建された火傷のマウスモデル

Mouse Wound Models and Preparation of Single-Cell Suspensions