Name: in vivo evaluation of fracture callus development during bone healing in mice using an mri-compatible osteosynthesis device for the mouse femur
Uploaded: 2017-11-14T15:00:00.000+00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur at JoVE.com

優れたテクニカル サポート、Sevil ニワトコヒゲナガアブラムシ、ステファニー Schroth、ベレナ フィッシャー、カーチャ Prystaz、イヴォンヌ Hägele とアン Subgang に感謝します。我々 はまた、ドイツ研究振興協会 (CRC1149, INST40/499-1) と本研究の資金の AO 外傷財団ドイツに感謝します。

すべての動物実験管理と実験動物の使用のための国際規則を遵守し、(第 1250, Regierungspräsidium テュービンゲン、ドイツ) は、地域の規制当局によって承認されました。すべてのマウスは 14 h ライトにケージごと 2 ~ 5 動物のグループで維持され、水と食料と 10 h 暗い概日リズム提供 広告自由。
 1 です。 手術材料やマウスの前処理の準備 <ol><li>定置滅菌すべて手術材料。滅菌時間の 20-30 分のための 120-135 ° C のオートクレーブ温度を使用します</li>。
	<li>購入 C57BL/6 マウスまたは 19 35 g 体重の間にある別の負担からマウス。適切な動物の世話や調査官によって承認されている国家のガイドラインに従って実験プロトコルに従う &#39; s 施設動物ケアおよび使用委員会。最低限の手順を開始する前に 7 日間馴化期間の許可します</li>。
	<li>術後 3 日まで術前に、1 日飲料水を介してすべてのマウスに鎮痛を提供します</li>。
</ol> 2。手術の手順と創外固定器の応用 <ol><li>管にマウスのプリロード 5-7% のイソフルランと 60 mL/分の酸素の場所。姿勢反射の損失の後麻酔誘導管から、マウスを削除し、1-3% のイソフルランと 60 mL/分の酸素を提供する吸入マスクで麻酔を維持します。
	麻酔中に <ol><li>モニター呼吸パターンと後肢足反射。呼吸数は約 100 サイクル/分、後肢の反射が存在しないことを確認します 。
		&#8203; 注: 必要なガスの量は年齢、性別、体重、およびマウスのひずみに依存します</li>。
	</ol></li> <li>術前注入マウス単一用量の抗生物質の皮下 (クリンダマイシン、45 mg/kg)。さらに、生体の体液バランスの維持のため 500 μ L の生理食塩水の皮下流体デポとマウスの挿入 (0.9 %nacl).</li>
	角膜を防ぐために <li>は、マウスの目に眼軟膏を適用、乾燥します。麻酔中に 37 ° C で加熱板の上にマウスを置くし、生理を維持する手術体温します</li>。
	<li>は右後肢から毛皮を取り外してアルコール ベースの消毒薬と外科の領域をスクラブします。無菌ではない区域を避けるために滅菌手袋の小さな部分と右後肢をカバーします。3 回右後肢を消毒します。外科領域を除いて全体のマウスに滅菌ドレープを配置します</li>。
	<li>は、メスで右大腿骨の前方の側面に沿って縦皮約 1 cm を切開します。マイクロのハサミ、鉗子で 大腿二頭筋筋 と 外側広筋 を露骨に区切ります。骨の前外側部分への無料アクセスを許可するようにマイクロ シザーと大腿骨転子で腱原点側をカットします。坐骨神経が保持されることを確認してください</li>。
	<li>は、大腿骨に平行、創外固定器 ( 図 1 A N/mm 3 の軸剛性) を配置します。手動で 0.45 mm のドリルビットと皮質を介して孔をドリルし、試錐孔にピンをマウント セラミックを配置します。最も遠位ピンに続いて、最も近位ピンと 2 つのピン間に起動します。
	<ol><li>がない引張、圧縮、またはせん断力、創固定器取付手順の中にそうでなければ骨切り達成のギャップは、創固定器の緩和のため十分なされませんを確認してください</li>。
	</ol></li> <li>鋸引きのプロシージャ中に脱水症状を避けるために滅菌食塩の量が少ないと骨を加湿します</li>。
	<li>0.4 mm のジリ ワイヤソーを用いた 2 つの内側のピン間全体の骨骨切り術 0.4 mm を作成します 。
	注: 必要に応じて、振動マイクロ見た使用できます、骨切り術を作成します。骨切り術領域で見たから任意の金属チップを避けるようにしてください</li>。
	<li>フラッシュ 2 つの骨折した皮質間骨チップを削除する滅菌食塩の 2 mL で慎重に骨切り術ギャップ</li>。
	<li>吸収性縫合糸の連続縫合を用いた筋肉の適応 (材料の表 を参照してください)。それから非吸収性縫合 (材料の表 を参照) を使用して、肌を調整します。噛み傷を避けるため、傷の頭蓋部に縫合線を配置しないでください 。
	注: は皮膚接着剤を使用しない、またはクリップ マウスは通常傷の原因からそれを削除するのでさらに肌にダメージを与える</li>。
	<li>消毒剤で外科の領域をきれいにし、そのケージ モニターにマウスにマウスを置くとそれまで供給十分な熱を (例えば 赤外光によって) は完全に目を覚まし。 手術後水、食物摂取量と体重を監視。動物は痛みや苦痛のないことを確認します。術後 3 日まで飲料水を介してすべてのマウスに鎮痛を提供します 。
	注: 最大 4 つの動物のグループで収容されるマウスします</li>。
	<li>監視マウス &#39; 手術後日 1 に 5 の活動。その経過中にマウスは作動肢に体重を支える必要があります。それ以外の場合、マウスはさらに解析から除外する必要があります</li>。
</ol> 3。Mri 検査の手順と画像解析 <ol><li>MRI スキャンのプロシージャ、する前に 2.1 と 2.3 の手順でプロトコルに従ってマウスを anaesthetize し、して、呼吸速度が約 100 サイクル/分の右後肢で創外固定器を挿入、カスタムメイドの取り付け装置 ( 図 1 B、C) に慎重にマウス。
	<ol><li>これは骨折治癒を妨げることがありますので、曲がるか、またはこの手順中に、創固定器の圧縮を避けることを確認します 。
		注: MRI スキャン早く動物のケアと実験的プロトコルに応じて、術後 3 日も実施できます</li>。
	</ol></li> <li>MRI 装置導入のための温度制御クレードル上にマウスを置きます。剛 4 要素の先頭のコイル マウント デバイスに接続します</li>。
	<li>11.7 t 営業専用高電界小動物 MRI システムを用いた MRI の取得データ 
	&#8203; 注: ネジに直交の大腿骨骨付き、MRI データ集録ジオメトリを配置します。
	プロトン密度脂肪抑制マルチ スライス東証シーケンス (PD 東証) を適用することによって <ol><li>取得データ集録パラメーターを使用して: エコー/繰り返し時間テ = 5.8 ms/TR 2,500 ミリ秒、解像度 Δr を = = 52 × 52 × 350 µm³ の視野 (FOV) =20 × 20 mm ²、および帯域幅 Δω = 150 KHz</li>。
		<li>注: 22 スライスの総収集時間は 36 分</li>
	</ol></li> <li>画像解析ソフトで取得したデータを開きます。0.35 mm 3 × 0.05 × 0.05 ボクセル サイズを入力します。次のように半自動しきい値設定法とその強度に基づく破壊カルス (骨、軟骨、線維性組織/骨髄) のさまざまな組織をセグメント化します。
	<ol><li>をクリックして、&#34; 新しいラベル フィールドの編集 &#34; をクリックして &#34; 追加材料 &#34; にマテリアルの名前を変更 &#34; カルス &#34;。使用して骨膜からハイポ強烈な信号に基づいて周囲の組織からカルス領域を区別、&#34; なげなわ &#34; ツール</li>。
		<li>クリック &#34; 材料に追加 &#34;。クリックして &#34; 追加材料 &#34; にマテリアルの名前を変更 &#34; 軟骨 &#34;。軟骨をセグメントを使用して、&#34; のしきい値 &#34; ツールと &#34; 現在材料のみを選択 &#34; から &#34; カルス &#34;。クリックして &#34; 軟骨 &#34;、&#34; 材料に追加 &#34;。これらの手順を繰り返します &#34; 骨 &#34; と &#34; 骨髄/線維組織 &#34; です</li>。
	</ol></li> <li>画像解析ソフトを利用したデータをセグメンテーションする組織に基づく骨折に対する大腿骨の 3 D 復元を生成します。クリックして &#34; 生成表面 &#34;、適用 &#34; なし &#34; の &#34; スムージング タイプ &#34; クリック &#34; 表面表示 &#34;. 
	注: 非常に小さな、超強烈な en の周辺骨折の野の ds は骨から軟部組織への移行のための成果物になる可能性があります。これらの分野は、さらなる分析から除外すべき。骨折治癒を表す軟骨組織の軟骨段階でカルスの破壊の途中でハイパー激しいエリア。軟骨内骨化段階で骨切り術ギャップから遠位骨折カルスでハイポ激しい分野と癒しの段階の後で全破壊カルス中で同じ強度のエリア新たに形成された骨カルス組織を表します。これらの領域には、ハイポ強烈な信号があるが、成熟した骨 (皮質) からの信号強度はさらに低いです。しきい値処理後は、骨組織と軟骨組織破壊カルスでの信号の強さは骨髄として残りの組織や線維組織をマークします。組織の分割の値: 3.4 5.4 範囲骨髄/線維組織、骨の組織 (成熟皮質、骨、骨カルス組織を含む) 1 3.3 (成熟皮質に正規化された信号強度) の範囲内でセグメント化と5.5 6.2 の範囲内軟骨カルス</li>。
	<li>、必要な場合は、MRI スキャンを骨折治癒経過中に縦繰り返します。軟骨のカルスの開発を監視し、手術後 10、14、21 日にマウスをスキャンする 。
	注: タイム ポイントは動物のケアと実験的プロトコルに依存可能性があります</li>。
</ol>

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著者のローマ人は、RISystem AG Davos、スイス連邦共和国、インプラントを生成し、この記事で使用される特定の楽器をインプラントの従業員です。他のすべての著者は競合する金融興味を持ってないです。

変更とトラブルシューティング:
本研究の主な目的は、軟骨骨折治癒プロセスの間に縦のカルス組織開発を監視する機能を持つマウスの大腿骨骨切り術、MRI 対応創外固定器の使用のためのプロトコルを記述するためだった。創外固定器の挿入のためのカスタムメイド取付デバイスのデザインは、繰り返しスキャン中に標準化された位置を確保しました。半自?...

二次癒合骨癒しの最も一般的な形式です。生育に伴う軟骨内骨化1,2,3の特定の側面を模倣した複雑なプロセスです。早期の骨折血腫は主に免疫細胞、粒状化および線維組織ので構成されます。低酸素分圧および高い生体力学的緊張破壊ギャップで骨芽細胞分化を妨げるが、軟骨4,5,6前駆細胞の分化を促進します。これらの細胞は、骨の骨折の初期安定性を提供する軟骨基質を形成する傷害の部位で増殖を開始します。カルスの成熟過程における軟骨細胞の肥大、なり受けるアポトーシス、またはトランス-骨芽細胞に分化します。軟骨の骨転移の地帯で血管新生は、7骨組織の形成を許可する高酸素レベルを提供します。骨破壊ギャップの橋渡し後、生体力学的安定性が増加し、生理学的な骨の輪郭と構造3を得るために発生する外部破壊カルスの培養破骨細胞を改造します。したがって、線維、軟骨、骨破壊カルス組織の量は骨治癒過程に関する重要な情報を提供します。カルス組織の発達人間とマウス8,9,10,11の両方の変化によって癒しの障害または遅延が表示されます。デジタル ラジオグラフィーとイメージング12,13µCT 縦カルス組織における前臨床骨折治癒の小動物を使用して研究を監視する生体内で利用できるテクニックが含まれます。ただし、両方の技術は無機非石灰化組織の間で区別することができるのみです。対照的に、MRI は優れた軟部組織コントラストを提供し、軟部組織や軟骨破壊のカルスを識別することがあります。
前作は、膜内骨欠損治癒15中マウスの死後MRI マウスにおける関節骨折14とin vivo MRI のための有望な結果を示した。しかし、両方の研究はまた限られた空間解像度と組織のコントラストを述べました。以前、マウス軟骨骨折16を治癒の間に柔らかいカルス形成の縦断的評価のため生体内で高分解能 MRI の可能性を示した。ここでは、MRI 対応に対して、創外マウスの大腿骨骨切り術を用いた軟骨骨折治癒経過中に縦カルス組織開発を監視するためにプロトコルを報告します。創外固定器の挿入のためのカスタムメイド取付デバイスのデザインは、繰り返しスキャン中に標準化された位置を確保しました。

<table><tbody><tr><td>Anaesthesia tube</td><td>FMI, Seeheim, Germany</td><td>ZUA-82-ANA-TUB-Mouse</td><td></td></tr><tr><td>Anaesthetic machine&amp;nbsp;</td><td>FMI, Seeheim, Germany</td><td>ZUA-82-GME-MA</td><td></td></tr><tr><td>Artery forceps&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BH104R</td><td></td></tr><tr><td>Autoclave</td><td>Systec, Wettenberg, Germany</td><td>DX-150</td><td></td></tr><tr><td>Autoclaving packaging</td><td>Stericlin, Feuchtwangen, Germany</td><td>2301-04/06/10/12/16</td><td></td></tr><tr><td>Avizo software</td><td>FEI, Burlington, USA</td><td>-</td><td>Version 8.0.1</td></tr><tr><td>BioSpec 117/16 magnetic resonance imaging system</td><td>Bruker Biospin, Ettlingen, Germany</td><td>117/16</td><td></td></tr><tr><td>Bulldog clamp&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BH 021R</td><td></td></tr><tr><td>Carbon steel scalpel no. 11/15</td><td>Aesculap, Tuttlingen, Germany</td><td>BA211/215</td><td></td></tr><tr><td>Ceramic mounting pin 0.45 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>HS691490</td><td></td></tr><tr><td>Clindamycin (300 mg / 2ml)</td><td>Ratiopharm, Ulm, Germany</td><td>-</td><td></td></tr><tr><td>Dressing forceps 115 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD210R</td><td></td></tr><tr><td>Dressing forceps 130 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD025R</td><td></td></tr><tr><td>Drill bit coated 0.45 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>HS820420</td><td></td></tr><tr><td>Durogrip needle holder 125 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BM024R</td><td></td></tr><tr><td>Foliodrape&amp;nbsp;</td><td>Hartmann, Heidenheim, Germany</td><td>2513026</td><td></td></tr><tr><td>Frekaderm</td><td>Fresenius, Bad Homburg, Germany</td><td>4928211</td><td></td></tr><tr><td>Gigli saw 0.44 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>RIS.590.110.25</td><td></td></tr><tr><td>Hand drill</td><td>RISystem, Davos, Switzerland</td><td>RIS.390.130-01</td><td></td></tr><tr><td>Heating plate&amp;nbsp;</td><td>FMI, Seeheim, Germany</td><td>IOW-3704</td><td></td></tr><tr><td>Hygonorm gloves&amp;nbsp;</td><td>Hygi, Telgte, Germany</td><td>2706</td><td></td></tr><tr><td>Isoflurane</td><td>Abbot, London, UK</td><td>Forene</td><td></td></tr><tr><td>Micro forceps 155 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD343R</td><td></td></tr><tr><td>Micro scissors 120 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>FD013R</td><td></td></tr><tr><td>Mouse FixEx L 0.7 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>RIS.611.300-10</td><td></td></tr><tr><td>Needle case for drills&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BL911R</td><td></td></tr><tr><td>Needle holder</td><td>Aesculap, Tuttlingen, Germany</td><td>BB078R</td><td></td></tr><tr><td>Octenisept</td><td>Sch&amp;uuml;lke, Norderstedt, Germany</td><td>121403</td><td></td></tr><tr><td>Osirix software</td><td>Pixmeo SARL, Bernex, Switzerland</td><td>-</td><td>Version 4.0</td></tr><tr><td>Oxygen, medical grade</td><td>MTI, Ulm, Germany</td><td>-</td><td></td></tr><tr><td>Resolon 5/0</td><td>Resorba, N&amp;uuml;rnberg, Germany</td><td>88143</td><td></td></tr><tr><td>Saline 0.9%</td><td>Braun, Melsungen, Germany</td><td>3570350</td><td></td></tr><tr><td>Scalpel handle 125 mm</td><td>Aesculap, Tuttlingen, Germany</td><td>BB073R</td><td></td></tr><tr><td>Scissors 150 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BC006R</td><td></td></tr><tr><td>Sealer for autoclave packaging&amp;nbsp;</td><td>Hawo GmbH, Obrigheim, Germany</td><td>HM500</td><td></td></tr><tr><td>Sterican 27 G&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>4657705</td><td></td></tr><tr><td>Sterile surgical blades no. 11/15&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BB511/515</td><td></td></tr><tr><td>Surgical gloves&amp;nbsp;</td><td>Hartmann, Heidenheim, Germany</td><td>Peha-micron 9425712</td><td></td></tr><tr><td>Surgical light&amp;nbsp;</td><td>Maquet SA, Ardon, France</td><td>Blue line 80</td><td></td></tr><tr><td>Syringes 5 ml&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>Injekt 4606051V</td><td></td></tr><tr><td>Tissue forceps 80 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>OC091R</td><td></td></tr><tr><td>Tramadol 25 mg/l</td><td>Gr&amp;uuml;nenthal, Aachen, Germany</td><td>100mg/ml</td><td></td></tr><tr><td>Vasofix Safety&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>4268113S-01</td><td></td></tr><tr><td>Vicryl 5-0&amp;nbsp;</td><td>Ethicon, Norderstedt, Germany</td><td>V30371</td><td></td></tr><tr><td>Visdisic eye ointment&amp;nbsp;</td><td>Bausch &amp;amp; Lomb, Berlin, Germany</td><td>3099559</td><td></td></tr></tbody></table>

in vivo evaluation of fracture callus development during bone healing in mice using an mri-compatible osteosynthesis device for the mouse femur

軟骨骨折治癒骨折カルス線維、骨、軟骨組織の開発を含む複雑なプロセスです。カルスのさまざまな組織の量は骨折の治癒経過に関する重要な情報を提供します。縦小動物を用いた前臨床骨折治癒研究におけるカルス組織の発達を監視する生体内で利用できる技術には、デジタル撮影と µCT イメージングが含まれます。しかし、両方の技法、石灰化と非石灰組織を区別することができるのみ。その結果、線維組織からの軟骨を区別するため不可能です。対照的に、磁気共鳴画像 (MRI) は含水率に基づく解剖学的構造を可視化して、軟部組織や軟骨破壊のカルスを非侵襲的識別することがあります。ここで、MRI 対応に対して、創外マウス大腿骨のマウスにおける骨再生中に MRI スキャンを許可するの使用を報告します。実験を創固定器とカスタムメイドのマウント デバイスできることを紹介反復的な mri 従って破壊カルス組織の発達に関する縦断的分析を有効にします。

最初に、(図 2の例を参照してください), MRI スキャンの解析による手術の成功を確認できます。すべての 4 つのピンは大腿骨骨幹部の真ん中に位置する必要があります。骨切り術のギャップのサイズは 0.3 から 0.5 の間する必要があります mm。骨切り術のギャップのサイズは、これらの値から大きく異なる場合、マウスをさらに分析から除外すべ?...

Watch this Scientific Journal Video about In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur at JoVE.com

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

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

軟骨内骨の治癒過程における破壊カルス組織の発達の評価は、癒しのプロセスを監視するため不可欠です。磁気共鳴画像 (MRI) の使用を報告する-マウスにおける骨再生中、MRI を許可するマウス大腿骨の互換性のある創の外固定が器にスキャンします。

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

in-vivo-evaluation-fracture-callus-development-during-bone-healing

Research

JoVE Journal

Medicine

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

9.5K ビュー.  University Medical Center Ulm. 軟骨内骨の治癒過程における破壊カルス組織の発達の評価は、癒しのプロセスを監視するため不可欠です。磁気共鳴画像 (MRI) の使用を報告する-マウスにおける骨再生中、MRI を許可するマウス大腿骨の互換性のある創の外固定が器にスキャンします。

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

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

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

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

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

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

医学

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

合わせた<em&gt;インビボ</em&gt;光とμCTイメージングマウスにおける整形外科インプラント感染で感染症、炎症、および骨解剖学を監視する

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

マウスにおけるペリオスチール骨格幹細胞のCCL5誘導性移行のリアルタイムイメージング

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

発生生物学

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

マウスにおける正常および不良骨治癒を分析するために大腿骨骨切り術の標準化された固定用髄内ロッキングネイル

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

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 ...

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 ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

安定化ピン付きマウス大腿骨の横骨折

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

生物学

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

マイクロCTを用いた骨折治癒の評価

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

マウスにおける骨幹大腿骨骨折モデルの確立

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

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

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