この研究は、NIDCR / NIHからR01DE023348（RHK）とカリフォルニア大学ロサンゼルス部門の学術上院の研究評議会から教員研究助成（RHK）によってサポートされていました。

マウスは、ジャクソン研究所から購入し、実験動物医学のUCLA課（DLAM）における病原体のない動物施設で飼育しました。実験は、学長の動物研究委員会（ARCの＃2016から037）から承認された機関のガイドラインに従って行いました。 1.マウスの麻酔<ol><li> 8週齢の雌C57 / BL6マウスを使用した（n = 3）。 </li><li>ケタミン（マウス体重の80から120ミリグラム/キログラム）/キシラジン（5 mgの/マウスの体重のkg）の溶液を用いてマウスを麻酔し、10 mLの/ kgの用量で腹腔内（ip）に投与します。 </li><li>ケタミンを準備します（80から120ミリグラム/キログラム）/キシラジン（5mg / kg）ソリューションおよび10 mLの/ kgの用量で腹腔内（ip）に、それらを管理します。 </li><li>マウスが完全につま先のピンチを実行することにより麻酔していることを確認します。 </li></ol> 2.パルプキャッピング手順<ol><li>マウスの口に口ホルダーを配置します。 </li><li>彼そのようなテーブルの上に口ホルダーを固定します広告が上向きにされています。 </li><li>最初の上顎臼歯が完全に表示されるように、口の上に顕微鏡（10倍）を配置します。 </li><li>パルプは透明象牙質を介して表示されるまで20万rpmで高速ハンドピースで1/4ラウンドバーを使用して、途中で歯のエナメル質の部分を削除します。バリでパルプを公開しないでください。 </li><li> ＃15歯内K-ファイル（150μmの直径）を使用して、象牙質を通して穿孔し、パルプを公開します。 注：象牙質の破片は、パルプ中に押し込まれないように特別な注意が必要です。これは、四半期ごとのK-ファイルを回転した後、K-ファイルアウトを引っ張ることによって回避することができます。 </li><li>製造元の指示に従って滅菌H 2 OでMTAを混ぜます。エクスプローラの先端で露出してパルプにMTAを提供し、配置します。穏やかなタッピングによって公開されたパルプにMTAをパックするために、紙ポイント（ファイン）の裏面を使用してください。紙点の厚い側は平坦であるため、でき露出されたパルプへのMTAの適切な凝縮のため。 </li><li>それだけで歯をカバーして35％リン酸エッチング液を配置することにより、15秒のための歯をエッチングします。それは歯肉組織を刺激することができるように、エッチング液の配置を制限するために特別な注意を払ってください。 注：エッチング液をシリンジに来て、歯科用接着剤は、歯の上にマイクロメカニカル結合を媒介することで流れることができるように、歯の表面を粗面化するために使用されます。それらは粘性であるため、それが歯に直接少量を適用することによって、自己完結型であることができます。 </li><li>エッチング液を除去するために、負の圧力をかけ吸引を使用してください。軽くエッチング剤の残留物を除去するために、H 2 Oで浸漬された綿ペレットを使用してください。エッチング液が歯から完全に除去されるまで、この手順を繰り返します。 </li><li>圧縮エアダスターを使用し、軽く歯を乾燥させます。 </li><li>紙ポイントの裏面を使用した歯科用接着剤を適用します。 </li><li> 3秒間の圧縮空気を使用して接着剤層を薄く。 </li><li> C言語URE硬化光ユニットを用いて20秒間の歯科用接着剤。 </li><li> MTAでキャップされた歯の上に少量で流動可能な複合材料を配置します。歯の溝に複合体を流れるようにエクスプローラの先端を使用してください。 </li><li>それを重合する光硬化ユニットを用いて30秒間複合体を硬化します。コンポジットが完全に硬化し、ハードエクスプローラを使用していることを確認してください。 </li></ol> 3.術後のケア<ol><li>すぐに覆髄手順の後にカルプロフェン（5 mgの/ kg）を皮下（SC）を管理します。 </li><li>彼らが目を覚ます前に暖かい動物を保つために低電力で加熱パッド上にマウスを置きます。 </li><li>住宅用ビバリウムにマウスを返します。 </li></ol> 4.組織調達<ol><li> 5の後 - 6週間、イソフルランとの完全な麻酔状態の下で頸椎脱臼によりマウスを安楽死させます。 </li><li>慎重に頭蓋底から上顎を除去し、50 mLチューブに入れて。 entirを修正しました。4℃で一晩、パルプでキャップされた歯とPBS、pH7.4中の4％パラホルムアルデヒドでキャップされていない反対側の歯の両方が含まれ、その後70％エタノール溶液に保存し、電子の上顎。 注：パラホルムアルデヒドは有毒で発癌性です。標準作業手順（SOP）に概説されるように適切な使用のパラホルムアルデヒドを監視する必要があります。 </li><li> μCTスキャンを用いてマウスの上顎をスキャンします。 70％エタノールを浸したガーゼでサンプルをラップし、15 mLの細胞培養チューブの中に置いて、スキャン中に上顎を固定します。 </li></ol> 5.μCTスキャン<ol><li> μCTスキャンのためのサンプルを準備します。簡潔には、70％エタノールに浸したガーゼで試料をラップし、一般的な15-mLの細胞培養コニカルチューブに固定します。メーカーの説明書で概説されるように、μCT走査ステージ上にチューブをマウントします。 </li><li> 145μAの電流55のkVpの電圧、及び2の露光時にX線源を設定します00ミリ秒。 </li><li> 20μmの解像度でと0.5ミリメートルのAlフィルタとμCTスキャナーで画像取得を実行します。 </li><li>画像を再構成し、それ11を視覚化します 。 </li><li> μCTスキャンが完了すると、2週間、5％EDTA、PBS中4％スクロース（pH7.4）で脱灰し始めます。 </li></ol> 6.組織処理と染色<ol><li>パラフィンで脱灰組織を埋め込みます。埋め込む前に、すぐに第一大臼歯の前方矢状カットを行うことで、上顎をトリミング。埋め込む一方で、第一大臼歯の縦断面が切断面となるように、下向きのこの面を配置します。 </li><li>ミクロトームを用いて、厚さ5μmのスライドを準備します。パルプキャップ領域は、通常、ランドマークとして使用することができるdistopalatal（DP）ルートと一致します。光学顕微鏡下で組織学を調べ、μCT画像を比較することにより、関心の正確な面積を決定します。 </li><li> H＆E染色、deparaのためffinize、キシレン（2回）と連続的に希釈したエタノール（100％エタノール2回、95％エタノール2倍、および70％エタノール1×）でスライドを再水和。 </li><li>水道水でスライドを洗浄します。 </li><li> 2.5分間ヘマトキシリン溶液で染色し、水道水ですすいでください。 </li><li> 1分間、95％エタノールでスライドを浸します。 </li><li> 1分間エオシン溶液で染色し、水道水ですすいでください。 </li><li>連続希釈したエタノール（70％エタノール1倍、95％エタノール2倍、および100％エタノール3×）、キシレン（3×）で脱水。 </li><li>マウントソリューションでスライドをマウントします。 </li></ol>

<ol>
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The authors have nothing to disclose.

現在、歯髄幹細胞（DPSCs）13の歯原性分化に歯科材料、足場、または成長因子のin vivoでの効果を検証するために利用可能ないくつかの異なる実験モデルがあります。これらのモデルは、腎カプセル、または足場14,15と免疫不全マウスにDPSCsの皮下移植などの臓器へのDPSCsの異所性自家移植が含まれます。しかしながら、これらの方法はDPSCsに対するそれらの歯原性効果は同所パルプ環境で行われていないという点で制限されています。一方、歯の上パルプ、パルプ・キャッピング手順に同所移植は大型動物16,17で使用されています。これらのモデルは同所環境で歯原性の可能性を評価する上で貴重であるが、それらのモデルの使用は、パルプ創傷治癒と修復象牙質形成に限定されたメカニズムの洞察を提供し、自然の中で主に観察されます。 </P&gt; 本稿では、マウスでは、パルプ・キャッピングを実行するための詳細な方法を提示します。このステップバイステップの手順では、μCTスキャンで分析し、修復象牙質形成のための組織サンプルを評価し、上顎を収穫、マウスを麻酔クラスI様キャビティを準備し、覆髄材を配置することを含みます。私たちの覆髄マウスモデルは、研究コミュニティで広く入手可能であるトランスジェニックやノックアウトマウスの使用を可能にすることによって、修復象牙質の文脈におけるin vivoでの歯髄創傷治癒の基本的な分子機構を調査するのに役立つであろう。 最近の研究では、象牙質形成が18,19に観察されたいくつかのマウスモデルを示しました。斉藤ら。反動はなく、修復、象牙質形成を刺激するパルプの露出のないクラスI様準備を、作成しました。反動象牙質と修復象牙質の両方が三次象牙質に分類されています、歯に以下の外部刺激を形成します。しかし、既存の象牙芽細胞によって形成される反動象牙質とは異なり、修復象牙質は、歯髄が露出し、象牙芽細胞層を20に違反しているとなりDPSCsとして象牙芽細胞様細胞によって形成されます。したがって、診療所における実際の覆髄手順を表すものではありません。別の研究では、ガラスアイオノマーは、パルプ露光19をキャップするために使用されました。しかしながら、臨床試験は、ガラスアイオノマーは慢性炎症ではなく、修復象牙質21に誘発されたことを示しました。この点で、我々のパルプキャッピングマウスモデルは、より良い患者の実際のパルプキャッピングの手順を表します。 我々が6週間以上後のマウスを収穫するとき、修復象牙質形成は髄室と根管（ 図4）全体で発生したことは注目に値します。このような観察は、我々は6月で修復象牙質形成を予想通り、かなり予想外であります覆髄材とパルプの間ction。しかし、パルプの強力な石灰化は、特に比較的若い患者22に、臨床で観察されます。本研究で用いた8週齢のマウスを、「青年」23であると考えられるので、これらのマウスは、まだかなりの歯原性の可能性を保有することにより、可能性が存在します。したがって、マウスでの修復象牙質形成の経年変化を調べるために価値があるだろう。 私たちの組織学的検査は、修復象牙質が明らかにパルプをかぶった歯に形成されたが、修復中の象牙細管（赤矢印）および骨細胞（黒矢印）の存在によって証明されるように、両方の象牙質および骨形成の特性は、あった、ことを明らかにしました象牙質（ 図5）。このような観察は、修復象牙質形成が局所的に象牙芽細胞様の歯髄幹細胞、ならびにinfiltratinが存在することによって誘導され得ることを示唆しています周囲の骨からグラム間葉系幹細胞。 酒石酸耐性酸性ホスファターゼ（TRAP）染色によって決定されるように象牙質形成細胞と比較して、我々は、パルプ内には象牙質再吸収細胞は認められなかった（データは示さず）。確かに、歯髄や根尖炎症は、歯の周りの骨表面上ではなく、として、まだ未知のメカニズム24による象牙質表面に破骨細胞形成を誘導します。注目すべきは、既存の象牙質および新たに形成された修復象牙質（ 図4）との間に明確な境界がありました。以前の研究では、同様の現象を実証しました。歯が破骨細胞の機能を阻害どちらも、ビスホスホネートまたは抗RANKL抗体の存在下で抽出された場合、既存の層板骨と新たに形成された網状骨25との間に明確な境界設定がありました。この概念は、さらにパルプ中の象牙質吸収細胞の不在をサポートしています。総称して、私たちの確立マウスモデルは、pだろうパルプ創傷治癒およびin vivoでの修復象牙質形成のメカニズムを検討するためのユニークな機会をrovide。 歯髄キャッピングマウスモデルには限界があります。ヒトとマウスの間の遺伝的メイクは明らかに異なっています。完全なゲノムは、ヒトおよびマウスにおいて配列決定されており、マウスとヒト26,27間のタンパク質コード領域で約85％の類似性が存在します。この考え方に沿って、それは動物でキャッピングパルプに関連する所見が必ずしも人間28のものを反映していないことが示唆されました。それにもかかわらず、動物モデルは広く、関節リウマチ29、全身性ショック31のための骨粗しょう症30、リポポリサッカライド（LPS）投与のためのovariactomy誘発性骨量減少、結紮のためのコラーゲン誘導関節炎などの生体内でのヒト疾患を再現するための研究コミュニティで使用されています歯周32の配置。このように、畝を覆髄SEモデルは、in vivoでのパルプ創傷治癒と修復象牙質形成の分子メカニズムを調べるために不可欠となります。それにもかかわらず、ちょうど他の動物モデルと同様に、覆髄マウスモデルからの所見の解釈および検証は慎重に評価する必要があります。 要約すると、現在の研究では、マウスにおいて、キャッピング成功したパルプを示しています。で基礎となるメカニズムを解明するために広く利用可能な遺伝子操作されたマウス系統を利用する1）機会を：それは提供するため、他の既知のモデルとは異なり、この覆髄マウスモデルは、パルプ再生の分野で非常に貴重な研究ツールと修復象牙質形成を提供します分子レベル2）経済的に効率的な方法は、サンプルサイズを増加させることによって、統計的に有意な結果を得ることができます。さらなる研究は、in vivoでの修復象牙質形成の客観的定量化を含め、修復象牙質形成の年齢依存的効果、evaluaを待ちます臨床的に利用可能な覆髄材の化、および適切な歯髄創傷治癒および修復象牙質の再生のために必要とされる分子決定の検証。

Dental caries are one of the most prevalent oral diseases and the leading cause of surgical interventions to dentitions in almost all individuals1,2. The prognosis of surgical interventions and restorations of a tooth largely depends upon proper pulpal response and successful wound healing. Indeed, dental caries that penetrate deeply through the enamel and dentin frequently lead to the exposure of the underlying pulp tissue that is often "capped" with dental materials, such as calcium hydroxide (Ca(OH)2) or hydraulic calcium-silicate cements (HCSCs), including mineral trioxide aggregates (MTA). The ultimate goal of such a pulp-capping procedure is to expedite pulpal wound healing by regenerating reparative dentin, a physical barrier that functions as a "biological seal" to protect the underlying pulp tissue and to increase the life expectancy of the tooth and the overall oral health. However, the underlying mechanism of pulpal wound healing and reparative dentin formation is not fully understood.
To better understand the mechanisms of pulpal wound healing and reparative dentin formation in vivo, several animals were previously used, including monkeys, dogs, and pigs3-5. Among them, rats are frequently used because they are relatively smaller in sizes compared to the other animals, but their teeth are large enough to perform direct pulp capping without any technical difficulties6-10. These animal models are ideal alternatives to human studies for examining pulpal responses and reparative dentin formation. However, their utilization is limited to observational studies at the cellular level, and they scarcely provide mechanistic insights during reparative dentin formation at the molecular level.
Recent technical advances in genetic engineering provided invaluable and indispensable research tools-mice that harbor a gene that is either overexpressed or deleted-that are instrumental to studying molecular mechanisms of human diseases in vivo. The numbers of different strains of transgenic or knockout mice that are strategically inducible in a cell-specific manner are continually growing in the scientific community. Therefore, examining pulpal wound healing and reparative dentin regeneration in these mice would greatly help to expedite our understanding of these processes at the molecular level. However, the use of mice is significantly dampened, as performing a pulp-capping procedure on a mouse tooth is technically challenging due to its miniature size. Here, we present our reproducible method of performing direct pulp capping in mice for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

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development of a direct pulp-capping model for the evaluation of pulpal wound healing and reparative dentin formation in mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

ここでは、マウスの歯にキャッピングパルプを実行するためにステップバイステップの手順を示しました。マウスでキャッピングパルプの重要な側面の一つは、適切な装置を持つことです。この点において、10Xパワー倍率で顕微鏡を有する（ 図1A）が不可欠です。歯のClass-I様準備を作成するには、20万回転（ 図1B）の電気高速ハンドピースに1/4ラウンドのバリを使用しました。あるいは、圧縮空気を使用するものを含む他のエンジンは、歯を調製するために使用することができます。 図2A-2Eは、パルプキャッピングを実行するための代表的な手順が示されています。クラスIのような製剤は、（ 図2B）を行いました。水スプレーは、手順の間にマウスを紛らすことがあるので、その使用は推奨されません。このため、優しくintermiで歯を調製することが不可欠ですttentは過熱を防ぐためにストローク。圧縮空気の使用は、冷却効果を提供することをお勧めします。これは、修復象牙質形成（ 図2C）でのデータの解釈を妨げる可能性歯内治療ファイルでパルプを露出させ、パルプに象牙質の破片をプッシュしていない注意してください。これは、圧縮空気を使用することによって回避することができます。同様に、MTAは、パルプにすぎ押さずに露出してパルプに配置する必要があります。 MTAの配置は、穏やかなタッピング動作（ 図2D）で紙点の裏面を使用することによって達成することができます。 MTA配置した後、歯は、歯の上に、複合体の結合を妨害する可能性がある、溝内に残っているMTAを除去するために、H 2 O-浸した綿ペレットを使用して洗浄する必要があります。複合修復する従来の方法は、（流動可能な複合材料を、歯の表面をエッチング接着剤でプライミングおよび結合し、配置して硬化を含め、使用されています図2E）。 6週間パルプキャッピング後、マウスを回収し、そして上面図を複合体は（ 図3A）依然として無傷であったことを確認するために撮影しました。 μCTスキャンは、修復象牙質が歯髄に形成されたことを示唆している、パルプをかぶったグループ（ 図3B）に歯髄スペースの大幅な景気後退を示しました。脱灰組織サンプルはさらに、生体内で組織学的に修復象牙質の形成を調べるためにH＆E染色に供しました。対照群では、象牙芽細胞層（OB）が象牙質（ - 4C図4A）のエッジの周りに顕著に明らかでした。対照的に、パルプキャッピング基は、歯髄空間（ - 4F図4D）に形成された修復象牙質のかなりの量（RD）を有していました。興味深いことに、綿密に検討を修復象牙質（RD）は象牙質の典型的な特徴（ 例えば、秒を示したことを明らかにしました<html>象牙細管、赤い矢印を表す線）、ならびに骨のトラップされた骨芽細胞、黒色の矢じりを表す（ 例えば、骨細胞）をtriated。私たちは象牙質マトリックスタンパク質1（DMP1）、歯原性分化12のマーカーについて染色したとき、我々はキャップされていない歯（ 図5Aおよび5B）に比べパルプに覆われた歯の歯髄におけるDMP1の発現の有意な増加を発見しました、修復象牙質の歯髄内に形成されたことを示します。 <img alt="図1" src="/files/ftp_upload/54973/54973fig1.jpg" /> 図1：パルプ・キャッピング手順のための機器のセットアップ。 （A）マウスの歯を可視化するための顕微鏡（10倍）。 （B）の高速ハンドピースとパルプを公開するクラスI製剤を調製するための電動モータエンジン。= &quot;_空白&quot;&gt;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。 <img alt="図2" src="/files/ftp_upload/54973/54973fig2.jpg" /> 図2：キャッピング手順で代表的な手順。 （A）マウスにおける上顎第一大臼歯に準備されていない歯。 （B）四半期ラウンドのバリを使用して、初期のエナメル除去。歯内のファイルを使用して、（C）パルプ暴露。露出されたパルプ中の（D）MTA配置。歯の上（E）複合復元配置。バーは500μmで表します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54973/54973fig2large.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> <img alt="図3" src="/files/ftp_upload/54973/54973fig3.jpg" /> 図3：臨床プレゼンテーションとμCTScanninマウスにおけるパルプ、蓋をして、キャップのない歯のグラム。 （A）パルプをかぶった歯（左）とキャップなし歯（右）上でマウス上顎の咬合ビュー。 （B）上顎の断面μCT画像。バーは500μmで表します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54973/54973fig3large.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> <img alt="図4" src="/files/ftp_upload/54973/54973fig4.jpg" /> 図4：in vivoでの修復象牙質形成の組織学的証拠。 100X、200X、および400Xでのマウスでキャップされていない上顎第一大臼歯の（AC）H＆E染色。 100X、200X、および400Xでのマウスでは、パルプをかぶった上顎第一大臼歯の（DE）H＆E染色。 RD =修復象牙質;黒矢じり=バーは100μmで（OB =象牙芽細胞層を表し、骨細胞;赤色の矢印=象牙細管）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54973/54973fig4large.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> <img alt="図5" src="/files/ftp_upload/54973/54973fig5.jpg" /> 図5：DMP1の免疫組織化学染色。 400Xでのマウスでキャップされていない上顎第一大臼歯の（A）DMP1染色。 400Xでのマウスでは、パルプをかぶった上顎第一大臼歯の（B）DMP1染色。バーは100μmで表します。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54973/54973fig5large.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 Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice at JoVE.com

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

我々は、in vivoでの歯髄創傷治癒と修復象牙質形成の評価のために、マウスの歯の上にキャッピング直接パルプを行うステップ・バイ・ステップ方式を説明します。

歯髄の評価のための直接覆髄モデルの開発、創傷治癒およびマウスにおける修復象牙質形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

development-direct-pulp-capping-model-for-evaluation-pulpal-wound

Nanyang Technological University

Research

JoVE Journal

Medicine

11.2K ビュー.  The UCLA School of Dentistry. 我々は、in vivoでの歯髄創傷治癒と修復象牙質形成の評価のために、マウスの歯の上にキャッピング直接パルプを行うステップ・バイ・ステップ方式を説明します。 

ビデオ: Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

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

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 ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

生体内で破壊カルス開発マウス大腿骨の骨、MRI 対応のデバイスを用いたマウスの骨治癒過程の評価

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

顕微鏡法によるIn Vitro創傷が治癒過程における上皮移行の評価

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

癒しの標準化された生体力学的条件下でマウスの軟骨破壊を分析する低侵襲モデル

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

マウスで閉じた大腿骨骨折の生成: 骨の治癒を研究するためのモデル

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

ターゲット灰白交通枝選択的化学腰椎交感神経切除術で

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...