We thank Amanda Fair, the CMI, and Kathy Sweet and the ORL at UM for assistance with &#181;CT imaging and analysis. Funding: BL Funded by 1K08GM109105-01 and Plastic Surgery Foundation National Endowment Award.

倫理文：実験動物研究所から第八版（ILAR、2011）とした：全ての動物手順は、実験動物の使用およびケアのためのガイドで提供されるガイドラインで定義されているように、良好な動物の練習に厳密に従って実施しましたミシガン大学（PRO0001553）の施設内動物管理使用委員会によって承認されました。 1.マウスの外科的処置注：使用8-10週齢のC57BL / 6マウス。他の年齢、背景、およびマウスの系統は、異なる条件または遺伝的構成をテストするために使用することができます。手続きの腱切除部分については、フェイスマスク、ヘアボンネット、および滅菌ガウン、手袋、および器具を使用して無菌状態を維持します。手術部位は無菌ポビドンヨード及び滅菌ドレープで準備する必要があります。獣医の加熱パッドを使用し、前administraに37℃に蘇生ソリューションを加温することにより低体温を避けまする。 <ol><li> 2.5％吸入イソフルランを用いてマウスを麻酔。簡単にアクセス背と後肢を残すためにノーズコーンを使用してください。粘膜の適切な呼吸数や着色を維持し、筋緊張、つま先のピンチ、および角膜反射をチェックすることにより、麻酔を確実にするために、イソフルランの投与速度を調整します。手順中にドライアイの怪我を防止するために、マウスの目に当たり障りのない眼軟膏を適用します。手術野の下の獣医の加熱パッドや温水循環装置を使用して、適切な体温を維持。 </li><li>鎮痛のための手術の皮下直前に、ブプレノルフィン、0.1ミリグラム/ kgで投与します。 </li><li>密接膝にかかとからバリカンを使用して、左後足に手術部位を剃ります。 </li><li>密接に背骨の上に始まり、背側正中線の左に剃毛領域を拡張する、火傷に対応するために、地域に少なくとも2cm×3cmをクリアし、マウスの背部を剃ります。 </li><li>3交流ポビドンヨードスクラブを使用して、膝に後足を含め、手術部位を準備します。 </li><li>左アキレス腱の内側面に沿って縦切開を行います。アキレス腱を容易に可視化することができるように、切開部を拡張します。約0.5センチメートル。 </li><li>シャープ組織ハサミで中点で腱の鋭い解剖とアキレス腱切除術を行います。腱の下に組織面での組織のはさみの刃を挿入し、ブレードが腱中間点になるまで平面に沿って詳細に分析。急激腱を切断するはさみの刃を閉じます。 注：腓腹筋、ヒラメ筋、および足底腱を含むすべての事後腱を含めることを確認してください。 </li><li>滅菌ガーゼで圧力をかけて止血を達成（最小限の出血があるはずです）と5-0ビクリルステッチで皮膚切開を閉じます。 </li><li>おおよそのmeasuremenで35グラムの重さのアルミニウムブロックと背部分層熱傷を行いますTS 2センチメートル×2センチ×3センチメートル17秒間マウスの剃毛背部に適用される水浴中で60℃に加熱しました。 注：ブロックの表面領域全体がマウスに接触している、しかし、ブロックに追加の圧力をかけることを避けることを確認して、麻酔したマウスの上にブロックを休んすることにより、適切な火傷の深さを実現、 すなわち、重力があることを可能にします所定の位置にブロックを保持する唯一の力。ブロックの一方の側に取り付けられた薄いプラスチック製のハンドルは、操作、安定化、および湯浴からの検索に便利です。これは、8〜10週齢のC57BL / 6マウスでは約30％、全体表面積の熱傷を作成します。この接触燃焼が原因で傷の上に火傷の深さの均一性と動物の間の再現に他の方法（火炎や熱傷熱傷）が選択されています。 </li><li>ガーゼで熱傷部位を乾燥さテガダームドレッシングを適用します。 </li><li>温め蘇生液を投与：乳酸リンゲル液1ミリリットルを腹腔内注射0.5mlの皮下注射。蘇生のこの1回投与は、火傷や腱切除からの回復のために十分です。 注：マウスを完全に回復するまでの監視の下できれいなケージに個別に、他のマウスと住宅ケージに家を麻酔したマウスを返さないでください。典型的な回復は、1-6時間以内に発生します。熱傷部位は、一般的に2〜4週間以内に治癒するときれいな住宅環境で守ればめったに創傷感染によって複雑にされていません。 </li><li>ブプレノルフィンを0.1mg / kgの皮下注射を手順、次の3日間ごとに12時間を管理します。適切な間隔でシリアルμCTスキャンとHOの成長を監視します。異所性骨の成長は、μCTによって術後3週間の周りに最初は明らかです。 注：HO開発の大部分は術後9週までに完了しています。私たちは繰り返しμCTとHOボリュームまたは場所の違いは、シングルエンドポイントは15週でスキャンμCTに比べて15週間にわたって隔週をスキャン確認されていません。実験の終了時に、施設のガイドラインに従ってCO 2吸入で全てのマウスを安楽死させると、10分後に頚椎脱臼で死亡を確認。 </li></ol> 2.μCT取得および分析<ol><li>腹臥位でスキャナベッドに麻酔をかけたマウスを固定します。呼吸モーションアーチファクトを防ぐために、しっかりと床に後肢をテープで固定します。イメージキャリブレーションのためのマウスの下にファントムを含む空気、水、およびヒドロキシアパタイトを含みます。 </li><li>骨解析ソフトウェアを開き、遠位後肢の先端に近位に股関節から後肢の両方を含む関心領域（ROI）を定義します。以下のパラメータを使用して、画像取得します。80 kVで、500μAと1300ミリ秒露光、48ミクロンのボクセルサイズを14,15 </li><li> 3ファントム室の各々にROIを描画するとの適切なフィールドに平均濃度を入力することにより、ハンスフィールド単位（HU）に画像のキャリブレーションソフトウェア。 </li><li>左後肢の脛骨が同所皮質骨構造の描写とHOのための明確な解剖学的ビューを可能にするために、Z軸に沿って平行になるように、ソフトウェアで「グラバー」ツールを使用して、再配向画像。 </li><li> HOが発生するまで、膝で始まり、画像スライスを通って遠位方向にスクロールします。マニュアルスプラインツールを使用して、足を介して、またはHOが突破されるまで、遠位に継続ごとに5 回目のスライス上に異所性骨の周りにROIを行います。すべてのHOを含むものROIにまとめたROIを拡張し、ステッチする外挿ツールを使用します。 </li><li> 3D ROIを作成し、分析メニューを選択します。最高の骨窓を示す下側および上側の閾値を設定することにより骨量を計算します。すべてのスキャンに同じ固定しきい値を使用してください。 </li></ol>

<ol><li>Leblanc, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BMP-9-induced+muscle+heterotopic+ossification+requires+changes+to+the+skeletal+muscle+microenvironment%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment.</a> J Bone Miner Res. 26 (6), 1166-1177 (2011).</li>
<li>Shore, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoinductive+signals+and+heterotopic+ossification%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">Osteoinductive signals and heterotopic ossification.</a> J Bone Miner Res. 26 (6), 1163-1165 (2011).</li>
<li>Wosczyna, M. N., Biswas, A. A., Cogswell, C. A., Goldhamer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multipotent+progenitors+resident+in+the+skeletal+muscle+interstitium+exhibit+robust+BMP-dependent+osteogenic+activity+and+mediate+heterotopic+ossification%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification.</a> J Bone Miner Res. 27 (5), 1004-1017 (2012).</li>
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<li>Yu, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BMP+type+I+receptor+inhibition+reduces+heterotopic+%5Bcorrected%5D+ossification%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">BMP type I receptor inhibition reduces heterotopic [corrected] ossification.</a> Nat Med. 14 (12), 1363-1369 (2008).</li>
<li>Culbert, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alk2+regulates+early+chondrogenic+fate+in+fibrodysplasia+ossificans+progressiva+heterotopic+endochondral+ossification%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Alk2 regulates early chondrogenic fate in fibrodysplasia ossificans progressiva heterotopic endochondral ossification.</a> Stem Cells. 32 (5), 1289-1300 (2014).</li>
<li>Dinther, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ALK2+R206H+mutation+linked+to+fibrodysplasia+ossificans+progressiva+confers+constitutive+activity+to+the+BMP+type+I+receptor+and+sensitizes+mesenchymal+cells+to+BMP-induced+osteoblast+differentiation+and+bone+formation%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation.</a> J Bone Miner Res. 25 (6), 1208-1215 (1359).</li>
<li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burn+injury+enhances+bone+formation+in+heterotopic+ossification+model%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Burn injury enhances bone formation in heterotopic ossification model.</a> Ann Surg. 259 (5), 993-998 (2014).</li>
<li>Scott, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brief+review+of+models+of+ectopic+bone+formation%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">Brief review of models of ectopic bone formation.</a> Stem Cells Dev. 21 (5), 655-667 (2012).</li>
<li>Tannous, O., Griffith, C., O'Toole, R. V., Pellegrini, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterotopic+ossification+after+extremity+blast+amputation+in+a+Sprague-Dawley+rat+animal+model%5BTitle%5D)+AND+%22J+Orthop+Trauma%22%5BJournal%5D)">Heterotopic ossification after extremity blast amputation in a Sprague-Dawley rat animal model.</a> J Orthop Trauma. 25 (8), 506-510 (2011).</li>
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<li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+of+heterotopic+ossification+through+remote+ATP+hydrolysis%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Treatment of heterotopic ossification through remote ATP hydrolysis.</a> Sci Transl Med. 6 (255), 255ra132(2014).</li>
<li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+detection+of+burn+induced+heterotopic+ossification+using+transcutaneous+Raman+spectroscopy%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy.</a> Bone. 54 (1), 28-34 (2013).</li>
<li>Perosky, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+detection+of+heterotopic+ossification+using+near-infrared+optical+imaging+reveals+dynamic+turnover+and+progression+of+mineralization+following+Achilles+tenotomy+and+burn+injury%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">Early detection of heterotopic ossification using near-infrared optical imaging reveals dynamic turnover and progression of mineralization following Achilles tenotomy and burn injury.</a> J Orthop Res. 32 (11), 1416-1423 (2014).</li>
<li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Aging+on+Osteogenic+Response+and+Heterotopic+Ossification+Following+Burn+Injury+in+Mice%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">Effects of Aging on Osteogenic Response and Heterotopic Ossification Following Burn Injury in Mice.</a> Stem Cells Dev. , (2014).</li>
<li>Alfieri, K. A., Forsberg, J. A., Potter, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blast+injuries+and+heterotopic+ossification%5BTitle%5D)+AND+%22Bone+and+Joint+Research%22%5BJournal%5D)">Blast injuries and heterotopic ossification.</a> Bone and Joint Research. 1 (8), 174-179 (2012).</li>
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</ol>

The authors have nothing to disclose

Heterotopic ossification represents a major functional impairment faced by patients that sustain trauma, burns, and invasive musculoskeletal procedures. The most at-risk population are soldiers in modern conflicts with major blast injuries from mechanisms such as improvised explosive devices (IED).18 Improved body armor and forward positioned medical units allows for improved survival of major extremity injury. After initial stabilization and repair of their extremity injury, these patients are at high risk of developing HO.18 Notwithstanding its prevalence and medical burden, relatively little is known about the details of HO development. Specifically, questions regarding the precise receptors and signaling pathways involved and the cell lineage responsible for HO are important current lines of inquiry. Some similarity to FOP and other forms of congenital HO are expected and supported by recent reports.8,14,17 A reliable model of endogenously produced HO in response to trauma is a key tool in this endeavor. 
Of particular interest is the development of novel diagnostic modalities that can detect where HO is forming at early stages when preventative treatment is viable. Current treatment modalities rely on surgical excision and radiation therapy which carry significant comorbidities and high rates of recurrence.5,19,20 This is largely due the fact that HO can only be identified after it has already formed and can then be detected by conventional imaging such as radiographs or CT scans. Spectroscopic studies including near-infrared imaging and Raman spectroscopy have shown some promising preliminary results but are still in developmental phases.15,16,21
One major advantage of the mouse model described herein is the reliability and predictability of where HO will form. This factor is key in the analysis of early HO development and the preceding events that allow for reactive bone formation. Because there is no preparation or application of exogenous substances, this protocol is relatively straightforward. Critical steps include maintaining sterile technique to avoid infection of the tenotomy site, ensuring uniform contact between the aluminum block and mouse skin while creating the contact burn, and ensuring that the Gastrocnemius, Soleus, and Plantaris tendons are severed when performing the tenotomy. While one advantage of this model is its ability to induce ectopic bone in response to trauma/burn only, it is also limited by excluding other injuries that cause of HO such as fractures, traumatic brain injury, and amputations. Also, while this model has been proven to be successful in mice, it has not yet been validated in larger animals that may more accurately mimic the amount of bone that is seen in human patients. 
Like all anabolic processes, ectopic bone formation is a multifactorial coordination between many different factors and players in the local environment and at the cellular level. It has been shown that HO develops in an endochondral process, however the specific precursor cell is still in question.8,14,17 Having a well-defined time-course and location of HO development is also useful in the trialing of potential preventative therapies for HO. Limiting bone morphogenetic protein (BMP) signaling or reducing inflammation following trauma/burn injuries with ATP hydrolysis have shown a significant reduction in reactive bone formation in this model.14 Further refinement and exploration of diagnostic and therapeutic modalities is certainly warranted. Key to that effort is a reliable mouse model of endogenously produced HO that mimics the processes that occur in patients that suffer the complications of HO.

Heterotopic ossification (HO) is the formation of ectopic bone in which osteo-potent cells are aberrantly induced to form endochondral bone outside of the skeleton. While the details of the HO formation pathway are still largely unknown, an accepted paradigm includes three key factors: an inflammatory inciting incident, a permissive niche, and mesenchymal stem cells capable of forming bone.1-3 HO is a common comorbidity complicating over 60% of major burn injuries, 65% of combat-related injuries, and 10% of invasive orthopaedic surgery cases.4,5 However, it is often difficult to predict where HO will form because it can occur at sites of local injury or at distant locations that may be otherwise uninjured. This variability in location makes it difficult to intervene prophylactically to prevent reactive bone formation in a locally targeted manner. There are also congenital forms of HO such as fibrodysplasia ossificans progressiva (FOP) in which patients are prone to the development of robust HO in response to minor trauma or inflammatory insult. Powerful animal models using transgenic mice have reproduced this phenotype and provided insight to the molecular pathways that may also be important in trauma induced HO.6-9 Translational research into the pathogenesis of non-congenital HO has used a wide variety of constructs ranging from injury alone to the implantation of exogenous osteo-inductive materials and/or cells.10-13
In our prior work we have validated a simple and reliable model of HO formation in mice which does not require the administration of any exogenous material.14-17 This model created two key conditions to initiate HO: local trauma and global inflammation. This was achieved through the use of an Achilles tenotomy (local trauma) combined with a distant burn injury (global inflammation). Mice received both treatments concurrently and were found to develop a robust amount of HO that could be analyzed by histologic, radiologic, and molecular means. Interestingly, concurrent burn injury significantly increased the amount of HO that formed and accelerated its developmental time-course.14-16 HO developed at predictable sites around the calcaneus, ankle joint, and tibia/fibula of the limb that received the tenotomy. The reliability of HO development at a known location allowed for focused examination of molecular and histologic features in the early stages of ectopic ossification.14,17 To date, 100% of mice (over 50 animals) with a tenotomy and concurrent burn injury have developed HO. Additionally, longitudinal 2D and 3D imaging and spectroscopic analysis were conducted to examine the growth pattern and biochemical make-up of HO.15,16

<table><tbody><tr><td>C57BL/6 mice</td><td>Jackson Laboratory</td><td>664</td><td>8-10 weeks old</td></tr><tr><td>Isoflurane &amp;ndash; Fluriso</td><td>VET one, Boise, ID</td><td>V1 501017</td><td></td></tr><tr><td>Buprenorphine &amp;ndash; Buprenex</td><td>Reckitt Benckiser Healthcare</td><td>NDC 12496-0757-1</td><td>0.3 mg/ml solution</td></tr><tr><td>Betadine</td><td>Owens and Minor, Mechanicsville, VA</td><td>2047PVP202</td><td></td></tr><tr><td>5-0 Vicryl sutures</td><td>Ethicon, Summerville, NJ</td><td>J493</td><td></td></tr><tr><td>Tegaderm Film, 6 cm x 7 cm</td><td>3M</td><td>1624W</td><td>Cut in half to properly cover burn site</td></tr><tr><td>&amp;micro;CT - GE eXplore Locus SP</td><td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td><td></td><td></td></tr><tr><td>Microview 2.2 Advanced Bone Analysis Application</td><td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td><td></td><td></td></tr></tbody></table>

direct mouse trauma/burn model of heterotopic ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

この本研究では、プロトコルは、外傷/火傷誘発されるHOの以前に公開されたマウスモデルに記載されている。14-17これはアキレス腱切除術と部分層熱傷を持つグローバル炎症侮辱と同時に、ローカライズ筋骨格傷害の作成 ​​が含まれます。これは、シリアル·イメージングで追跡することができる腱切除部位での反応性骨を確実に形成されます。現在までに、アキレス腱切除術の両方を持続し、熱傷したすべてのマウス（50以上）が腱切除部位でHOの測定可能な量を開発しました。異所性骨の形成を定量化するために、キャリブレーション画像μCTプロトコルは、所望の各時点で使用されています。アキレス腱切除術と組み合わせて、火傷の添加は重要なHOの誘導に​​おいて重要であることが証明されました。両方の怪我が同時に起こっての交流に比べ腱切除部位に形成された異所性骨の量の有意な増加がありますhillesの腱切除単独（ 図1）。17 <img alt="図1" src="/files/ftp_upload/52880/52880fig1.jpg" /> 同時熱傷を伴うまたは伴わないアキレス腱切除術、次の図1のHO開発を。全てのマウスは、左後肢のアキレス腱切除術を受けたマウスの半分はまた背部の30％TBSA部分層熱傷を受けました。 μCTスキャンは5日で完了し、3、5、7、9、および15週目には、損傷を投稿しました。 （A）。代表的な3次元再構成は、同所性骨白とHOが青色で表示されます。赤い丸は、HOのnidusesを示します。異所性骨は、腱切除の面積だけでなく、下肢における損傷部位への遠位端と近位の場所で開発しています。 clnically見HOと同様に、この反応性骨が骨格の骨と連続しているか、軟部組織でリモートで開発することができます。 （B）。 HOの定量化は、COMました較正済み画像プロトコルでpletedグラフに示します。 HOは、15週間を超えて、典型的には5-9週間の間に見られる最大の成長速度で9-15週間の間しかし、開発プラトーを持続しています。データは、平均+/- SD、群あたりn = 4、スチューデントの t検定、* P &lt;0.05であります （これは以前に17から変更されたデータを、公開されていることに注意してください）。 <a href="https://www.jove.com/files/ftp_upload/52880/52880fig1large.jpg" target="_blank">この図の拡大版を表示するには、こちらをクリックしてください。</a>

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Direct Mouse Trauma/Burn Model of Heterotopic Ossification

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

直接マウストラウマ/異所性骨化のモデルバーン

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

direct-mouse-traumaburn-model-of-heterotopic-ossification

Universidad San Sebastian

Research

JoVE Journal

Medicine

10.1K ビュー.  University of Michigan Medical School. An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

ビデオ: Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

偽骨折：急性末梢組織外傷モデル

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

医学

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

同所性骨肉腫および肺転移マウスモデルを生成するための脛骨内骨肉腫細胞注射

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

がん研究

A Mouse Distraction Osteogenesis Model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

マウス気晴らし骨形成モデル

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

発生生物学

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成体マウスの解離と再生:哺乳類のブラステマ形成と腸内形成を調べるための簡単なモデル

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

A Simple Critical-sized Femoral Defect Model in Mice

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

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

マウスの簡単な重要なサイズの大腿欠陥モデル

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

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

整形外科術後の高次脳機能障害と組織再生を研究するマウス モデル

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

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 ...

生物工学

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

<meta charset="utf8"></head><body>脛骨骨折ピンモデル:整形外科損傷の臨床的に関連性のあるマウスモデル

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

脛骨骨折ピンモデル:整形外科損傷の臨床的に関連性のあるマウスモデル

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

神経科学

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 ...

直接マウストラウマ/異所性骨化のモデルバーン

要約

さらに動画を探す

偽骨折：急性末梢組織外傷モデル

同所性骨肉腫および肺転移マウスモデルを生成するための脛骨内骨肉腫細胞注射

マウス気晴らし骨形成モデル

成体マウスの解離と再生:哺乳類のブラステマ形成と腸内形成を調べるための簡単なモデル

マウスの簡単な重要なサイズの大腿欠陥モデル

整形外科術後の高次脳機能障害と組織再生を研究するマウスモデル

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

脛骨骨折ピンモデル:整形外科損傷の臨床的に関連性のあるマウスモデル

脛骨骨折ピンモデル:整形外科損傷の臨床的に関連性のあるマウスモデル

同種異系皮膚移植片で再建された火傷のマウスモデル

直接マウストラウマ/異所性骨化のモデルバーン

要約

さらに動画を探す

偽骨折：急性末梢組織外傷モデル

同所性骨肉腫および肺転移マウスモデルを生成するための脛骨内骨肉腫細胞注射

マウス気晴らし骨形成モデル

成体マウスの解離と再生:哺乳類のブラステマ形成と腸内形成を調べるための簡単なモデル

マウスの簡単な重要なサイズの大腿欠陥モデル

整形外科術後の高次脳機能障害と組織再生を研究するマウス モデル

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

脛骨骨折ピンモデル:整形外科損傷の臨床的に関連性のあるマウスモデル

脛骨骨折ピンモデル:整形外科損傷の臨床的に関連性のあるマウスモデル

同種異系皮膚移植片で再建された火傷のマウスモデル

整形外科術後の高次脳機能障害と組織再生を研究するマウスモデル