Sevil Essig, Stefanie Schroth, Verena Fischer, Katja Prystaz, Yvonne Hägele ve Anne Subgang mükemmel teknik destek için teşekkür ederiz. Ayrıca bu çalışmada finansmanı için AO travma Vakfı Almanya ve Alman Araştırma Vakfı (CRC1149, INST40/499-1) teşekkür ederiz.

tüm hayvan deneyleri için bakım ve kullanım laboratuvar hayvanlarının uluslararası düzenlemelere uyulması ve bölgesel düzenleyici otoriteler tarafından (No 1250, Regierungspräsidium Tübingen, Almanya) kabul edildi. Bütün fareler yapılmaktadır 14-h ateşin üzerinde kafes başına iki ila beş hayvanların gruplarındaki 10-h karanlık sirkadiyen ritim su ve yiyecek ile sağlanan ad libitum.
 1. cerrahi malzeme ve farelerin ön arıtma hazırlanması <ol><li>Sterilize tüm cerrahi malzeme. 120-135 ° C bir ısıyla sıcaklık sterilizasyon zaman için 20-30 dk kullanın.</li>
	<li>Satınalma C57BL/6 fare veya başka bir zorlanma arasında 19-35 g vücut ağırlığı olan fareler. Uygun hayvan bakımı ve araştırmacı tarafından onaylanmış Ulusal kurallara uygun olarak deneysel iletişim kuralları izleyin &#39; s kurumsal hayvan bakım ve kullanım Komitesi. 7 gün iklimlendirme süre yordamına başlamadan önce en az izin.</li>
	<li>Bir gün önce ameliyat üçüncü ameliyat sonrası güne kadar içme suyu ile tüm fareler analjezi sağlamak.</li>
</ol> 2. Cerrahi prosedür ve eksternal fiksatör uygulaması <ol><li>bir tüp içine fare preloaded 5-%7 isoflurane ve 60 mL/dak oksijen ile yer. Postürel refleks kaybı sonra fareyi anestezi indüksiyon tüpünden kaldırmak ve % 1-3 isoflurane ve 60 mL/dak oksijen sağlayan bir solunum maskesi ile anestezi korumak.
	Anestezi sırasında <ol><li>monitör nefes desen ve hind pençe refleks. Solunum hızı yaklaşık 100 devir/dk ve arka pençe refleks yoktur olun. 
		&#8203; Not: yaş, cinsiyet, vücut ağırlığı ve zorlanma fare üzerinde gerekli gaz miktarını bağlıdır.</li>
	</ol></li> <li>Ameliyat öncesi, fareyi tek doz antibiyotik subkutan enjekte (Klindamisin, 45 mg/kg). Ayrıca, fizyolojik sıvı dengesi bakımı için fareyi 500 µL serum fizyolojik bir subkutan sıvı depo ile enjekte (% 0.9 NaCl).</li>
	Kornea önlemek için <li>kurutma, göz merhem fare gözler için geçerlidir. Anestezi sırasında bir ısıtma yüzeyi 37 ° C'de fare yerleştirin ve fizyolojik korumak için cerrahi müdahale vücut sıcaklığı.</li>
	<li>Kürk sağ arka bacak kaldırma ve cerrahi alan bir alkol bazlı dezenfektan ile fırçalayın. Sağ arka pençe ile hayır alanları önlemek için steril eldiven küçük bir bölümünü kapsar. Sağ arka bacak üç kez dezenfekte. Steril örtü cerrahi alan hariç tüm fare yerleştirin.</li>
	<li>Cilt yaklaşık 1 cm boyuna bir neşter ile sağ uyluk ön tarafındaki deşmek. Açık açık m. pazı kemiği ve m. vastus kasılmasına mikro makas ve Forseps ile ayırın. Ücretsiz kemiğin anterolateral parçası erişmesine izin vermek için mikro makas ile uyluk trochanter, tendon kökenli yan kesti. Siyatik sinir korunur emin olun.</li>
	<li>Eksternal fiksatör (3 N/mm, şekil 1 A eksenel sertlik) uyluk için paralel getirin. El ile 0,45 mm matkap ile korteks ile sondaj matkap ve iğne sondaj montaj seramik yer. En distal pim, ardından en proksimal PIN ve iki iğne ile arasında başlar.
	<ol><li>Hiçbir gerginlik, sıkıştırma veya yamultma stres fiksatör montaj prosedürü sırasında aksi takdirde elde osteotomi boşluğu fiksatör gevşeme nedeniyle yeterli olmayacaktır emin olun.</li>
	</ol></li> <li>Steril NaCl kesim işlemi sırasında kurutma önlemek için küçük bir miktar ile kemik nemlendirmek.</li>
	<li>Oluşturmak 0.4 mm gigli tel testere kullanarak iki iç iğne arasındaki bütün kemik üzerinden 0,4 mm osteotomi. 
	Not: İsteğe bağlı olarak, salınan bir mikrop saw osteotomi oluşturmak için kullanılabilir. Herhangi bir metal cips osteotomi alanda testere gelen önlemek emin olun.</li>
	<li>Osteotomi boşluğu dikkatle 2 mL kemik parçası getirdi iki kırık cortices arasında kaldırmak için steril NaCl ile flush.</li>
	<li>Uyum kasları tekrar Emilebilen dikiş ile sürekli dikiş kullanarak (Tablo malzemeleri görmek). Sonra cilt kesintiye uğramış-tekrar Emilebilen dikiş (Tablo malzemeleri görmek) kullanarak uyum. Yara ısırma önlemek için dikiş yara kafatası kısmında koymayın. 
	Not: deri yapıştırıcı kullanın veya fareler genellikle yara neden kaldırmak bu yana klipleri daha fazla zarar cilde.</li>
	<li>Cerrahi alan bir dezenfektan ile temiz ve fareyi onun kafes. monitör fare yerleştirin ve tedarik yeterli ısı (örneğin kızılötesi ışık tarafından) bu kadar tam olarak uyanık. izlemek su, gıda alımı ve vücut ağırlığı için ameliyattan sonra Hayvan acı ve sıkıntı içinde olmadığından emin olun. Üçüncü ameliyat sonrası güne kadar içme suyu ile tüm fareler analjezi sağlamak. 
	Not: Fareler dört hayvan gruplarında yer alan.</li>
	<li>İzlemek fare &#39; ameliyat sonrası 1-5 gün faaliyete s. O zaman ders sırasında fare ağırlık işletilen bacak üzerinde ayı olmalıdır. Aksi takdirde, fare daha fazla analiz dışlanması gerekir.</li>
</ol> 3. MRI yordam ve görüntü analizi <ol><li>önceki yordamı, tarama MRG için fareyi adım 2.1 ve 2.3 protokolüne göre uyuşturan ve solunum hızı 100 civarında tutmak devir/dak. doğru arka bacak eksternal fiksatör Ekle fare dikkatle içine bir ısmarlama montaj aygıt ( şekil 1 B, C).
	<ol><li>Bu kırık iyileşmesi ile girişime neden olabilir beri bükme veya fiksatör sıkıştırma bu adımı sırasında önlemek emin olun. 
		Not: MRI taramaları gibi erken 3 gün sonra cerrahi, hayvan bakımı ve deneysel protokol bağlı olarak yapılabilir.</li>
	</ol></li> <li>Bir sıcaklık kontrollü beşik için giriş içine MRI aygıt üzerinde fareyi getirin. Montaj aygıtlarını katı dört elemanlı baş bobinine bağlamak.</li>
	<li>11.7 T. çalışan özel bir yüksek-alan küçük hayvan MRI sistemi kullanarak elde MRG veri 
	&#8203; Not: MRG veri edinme geometri uyluk kemiği, uygun vidaları ile uyumlu.
	Bir proton-yoğunluk yağ bastırılmış çok dilim TSE sıra (PD-TSE) uygulayarak <ol><li>edinme veri toplama parametrelerini kullanarak: yankı/tekrarlama saat TE 5.8 ms/TR = 2500 ms, çözünürlük Δr = 52 × 52 × 350 µm³, görüş alanı (FOV) = = 20 × 20 mm ² ve bant genişliği Δω = 150 KHz.</li>
		<li>Not: 36 dk. 22 dilimleri toplam edinme zamanı</li>
	</ol></li> <li>Elde edilen veri görüntü analiz yazılımı ile açın. 0.05 0.05 0.35 mm 3 x x olarak Voksel boyutu girin. Aşağıdaki gibi onların şiddette, yarı otomatik eşik dayalı kırık Kallus (kemik, kıkırdak, fibröz doku/kemik iliği) farklı dokularda kesiminde.
	<ol><li>Tıklayın &#34; yeni etiket alanı Düzenle &#34;,'ı tıklatın &#34; malzeme eklemek &#34; ve malzeme için yeniden adlandırmak &#34; Nasıra &#34;. Kallus alan kapaklarini kullanarak hipo-yoğun sinyal temel çevre dokular ayırmak &#34; Kement &#34; aracı.</li>
		<li>Tıklama &#34; malzeme eklemek &#34;. ' I tıklatın &#34; malzeme eklemek &#34; ve malzeme için yeniden adlandırmak &#34; kıkırdak &#34;. Kıkırdak kullanarak segment &#34; eşik &#34; aracı ve &#34; sadece geçerli malzeme seçin &#34; üzerinden &#34; Nasıra &#34;. ' I tıklatın &#34; kıkırdak &#34; ve &#34; malzeme eklemek &#34;. Bu adımları yineleyin &#34; kemik &#34; ve &#34; kemik iliği/fibröz doku &#34;.</li>
	</ol></li> <li>Görüntü analiz yazılımı kullanarak doku segmentasyon verilere göre kırık uyluk 3B rekonstrüksiyonlar oluşturmak. ' I tıklatın &#34; yüzey oluşturmak &#34;, uygulamak &#34; hiç &#34; için &#34; yumuşatma türü &#34; ve'ı tıklatın &#34; yüzey görünümü &#34;. 
	Not: tr çevreleyen çok küçük, hiper-yoğun alanlarkırık cortices DS eserler kemikli yumuşak doku geçiş nedeniyle olması muhtemeldir. Bu alanlarda daha fazla analiz dışlanmaları gerekir. Kırık ortasında Hyper-yoğun alanlarda temsil kıkırdak doku şifa kırık endochondral aşamasında Nasıra. Osteotomi boşluğu endochondral ossifikasyon faz üzerinden distal kırık Kallus hipo-yoğun alanlarda ve alanları daha sonra aşamaları şifa, bütün kırık Kallus boyunca aynı yoğunluğu ile yeni kurulan kemik Kallus dokusu temsil eder. Her ne kadar bu alanlarda hipo-yoğun bir sinyal, olgun kemik (korteks) üzerinden sinyal şiddeti daha düşük olduğunu. Eşik sonra sinyal yoğunluğu kemik doku ve kırık Kallus kıkırdak doku, kemik iliği olarak kalan doku ve fibröz doku işaretleyin. Doku bölümleme değerleri: kemik doku (olgun korteks, Trabeküler kemik ve kemik Kallus dokusu dahil) 3.4-5,4, menzili içinde kemik iliği/fibröz doku 1-3.3 (olgun korteks için normalleştirilmiş sinyal yoğunluğu), Aralık içinde parçalara ve kıkırdak Kallus dokusu 5.5-6,2 aralığında.</li>
	<li>Gerekirse, MRI tarama boyuna süreci şifa kırık sırasında yineleyin. Kıkırdak Kallus gelişimi izlemek için 10, 14 ve 21 gün ameliyat sonrası fareler tarama. 
	Not: Zaman Puan hayvan bakımı ve deneysel iletişim kuralına bağlı olabilir.</li>
</ol>

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Romano Matthys yazar RISystem AG Davos, İsviçre implantlar üretir ve özel aletler bu makalede kullanılan implant bir çalışandır. Diğer yazarlar hiçbir rakip mali ilgi alanlarına sahip.

Değişiklikler ve sorun giderme:
Kallus dokusu geliştirme boyuna endochondral kırık iyileşmesi işlemi sırasında izleme olanağı ile uyluk osteotomi fare için bir MRI uyumlu eksternal fiksatör ile kullanmak için bir iletişim kuralı tanımlamak için bu çalışmanın ana hedefi oldu. Eksternal fiksatör yerleştirilmesi için özel montaj aygıtının tasarım standart bir pozisyon tekrarlanan taramalar sırasında sağlamıştır. Yarı otomatik doku segmentasyon...

İkincil kırık iyileşmesi kemik iyileşme en yaygın şeklidir. Ontogenic endochondral ossifikasyon1,2,3, belirli özellikleriyle taklit karmaşık bir süreçtir. Erken kırık hematom ağırlıklı olarak bağışıklık hücreleri, granülasyon ve fibröz doku oluşur. Düşük oksijen gerilim ve yüksek biyomekanik suşları osteoblast farklılaşma kırık boşluğu, engel ama progenitör hücre farklılaşma kondrosit4,5,6içine teşvik. Bu hücreler kırık kemiğin ilk istikrar sağlayan bir kıkırdak matris oluşturmak için yaralanma sitesinde çoğalırlar başlar. Kallus olgunlaşma sırasında kondrosit Hipertrofik, haline geçmesi apoptosis veya trans-dokusunu ayırt etmek. Neovaskülarizasyon kıkırdak-kemik geçiş bölgesi, yüksek oksijen düzeyleri, kemik doku7oluşumunu sağlayarak verir. Kemik kırığı köprü sonra daha biyomekanik istikrarlı ve fizyolojik kemik kontur ve yapısı3kazanmak için sebep harici kırığı Kallus remodeling oluşur. Bu nedenle, kırık Kallus fibröz kıkırdak ve kemik dokusunda miktarda kemik iyileşme süreci hakkında önemli bilgiler sağlar. Rahatsız veya gecikmeli şifa değişiklikler Kallus dokusu gelişme hem insanlar ve fareler8,9,10,11tarafından görünür hale gelir. Dijital Radyografi ve12,13görüntüleme µCT boyuna preklinik kırık küçük hayvanlar kullanarak çalışmalar şifa Nasırlaşma doku gelişiminde izlemek için kullanılabilir vivo içinde teknikleri içerir. Ancak, her iki teknikleri sadece mineralize ve mineralize olmayan doku arasında ayırımcılık edebiliyoruz. Buna ek olarak, Mr mükemmel yumuşak doku kontrast sağlar ve bu nedenle yumuşak doku ve kıkırdak içinde kırık Kallus tanımlamak mümkün olabilir.
Önceki çalışma Intramembranous kemik-kusur şifa15sırasında farelerde post mortem MRI farelerde ile eklem kırıkları14 ve in vivo MRI için umut verici sonuçlar gösterdi. Bununla birlikte, her iki çalışmaları da sınırlı uzamsal çözünürlük ve doku kontrast belirtti. Biz daha önce yüksek çözünürlüklü vivo içinde Mr16şifa fare endochondral kırık sırasında yumuşak Kallus oluşumu boyuna değerlendirmesi için fizibilite gösterdi. Burada, Kallus dokusu geliştirme boyuna endochondral kırık iyileşme süreci sırasında izlemek için uyluk osteotomi farelerde için bir MRI uyumlu eksternal fiksatör kullanarak için protokol raporu. Eksternal fiksatör yerleştirilmesi için özel montaj aygıtının tasarım standart bir pozisyon tekrarlanan taramalar sırasında sağlamıştır.

<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

Endochondral kırık iyileşmesi fibröz kıkırdak ve kemik dokusunda kırık Kallus gelişimi içeren karmaşık bir işlemdir. Kallus farklı dokularda miktarda ilerleme şifa kırık önemli bilgiler sağlar. Dijital Radyografi ve µCT görüntüleme boyuna preklinik kırık iyileşmesi çalışmalar küçük hayvanlar kullanarak Kallus dokusu gelişiminde izlemek için kullanılabilir vivo içinde teknikleri içerir. Ancak, her iki teknikleri sadece mineralize ve mineralize olmayan doku arasında ayırt edebiliyoruz. Sonuç olarak, kıkırdak fibröz doku ayırt etmek mümkün değildir. Buna ek olarak, manyetik rezonans görüntüleme (MRG) su içeriğine göre anatomik yapıları görüntüler ve bu nedenle Noninvazif yumuşak doku ve kıkırdak içinde kırık Kallus tanımlamak mümkün olabilir. Burada, Mr uyumlu fare uyluk için fiksatör MRI taramaları farelerde kemik yeniden oluşturma işlemi sırasında izin vermek için dış kullanımı raporu Deneyler fiksatör ve ısmarlama montaj aygıt tekrarlayan MRI taramaları, böylece kırık-Kallus dokusu gelişimin boyuna analizi etkinleştirmek izin gösterdi.

İlk olarak, cerrahi işlem başarısı analizi ( Şekil 2' deki örneğe bakın) MRI taramaları tarafından onaylanabilir. Tüm dört iğne ortasında femur şaft yerleştirilmemesi gerekir. Osteotomi boşluk boyutu 0.3-0.5 arasında olmalıdır mm. Osteotomi boşluk boyutunu büyük ölçüde bu değerler değişiyorsa, fare daha fazla analiz çıkarılmalıdır.
İkinci olarak, boyuna taramalar?...

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

Vivo Fare uyluk için bir MRI uyumlu Osteosentez aygıtı kullanma farelerde kemik iyileşme sırasında kırık Kallus gelişimi değerlendirme

Endochondral kemik iyileşme sırasında kırık Kallus dokusu geliştirme değerlendirilmesi iyileşme süreci izlemek için önemlidir. Burada, bir manyetik rezonans görüntüleme (MRG) kullanımı raporu-uyumlu eksternal fiksatör fare uyluk için MRG izin farelerde kemik rejenerasyon sırasında tarar.

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 Görüntüleme.  University Medical Center Ulm. Endochondral kemik iyileşme sırasında kırık Kallus dokusu geliştirme değerlendirilmesi iyileşme süreci izlemek için önemlidir. Burada, bir manyetik rezonans görüntüleme (MRG) kullanımı raporu-uyumlu eksternal fiksatör fare uyluk için MRG izin farelerde kemik rejenerasyon sırasında tarar.

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

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.

Ardışık<em&gt; In vivo</em&gt; Kırık Onarım sırasında Osteojenik Kök / Progenitör Hücrelerinin Görüntüleme

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

Tıp

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.

Kombine<em&gt; In vivo</em&gt; Optik ve uCT Görüntüleme Farelerde bir Ortopedik İmplant Enfeksiyon Enfeksiyon, Enflamasyon ve Kemik Anatomisi Monitör

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

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Farelerde bir Segmental Femoral Kritik boyutlu Defekt Modelinde kurulması Plaka Osteosentez tarafından Stabilize

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

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.

Femur Osteotomiler standardize Sabitleme için bir intramedüller Kilitleme Nail Fare Normal ve Arızalı Kemik Şifa Analiz

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.

Endochondral standart biyomekanik koşullar altında farelerde şifa kırık çözümlemek için minimal invaziv bir Model

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.

Farelerde kapalı femur kırığı nesil: kemik şifa çalışması için bir Model

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

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

Wistar Albino Sıçanlarında Femur Osteotomi Modeli Geliştirme Protokolü

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

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.

Fare Femurunun Stabilize Pin ile Enine Kırılması

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 ...

Biyoloji

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.

Kemik Kırığı İyileşmesinin Mikro Bilgisayarlı Tomografi Kullanılarak Değerlendirilmesi

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.

Farelerde Diafiz Femur Kırığı Modelinin Kurulması

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

Vivo Fare uyluk için bir MRI uyumlu Osteosentez aygıtı kullanma farelerde kemik iyileşme sırasında kırık Kallus gelişimi değerlendirme

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