Questo lavoro è stato supportato da RISystem AG, Davos, Svizzera.

Tutte le procedure sono state eseguite secondo le direttive del National Institutes of Health per l'uso di animali da esperimento e seguivano le linee guida istituzionali (Landesamt für Verbraucherschutz, Zentralstelle Amtstierärztlicher Dienst, Saarbrücken, Germania).
1. preparazione di strumenti chirurgici e protesi
<ol><li>Selezionare una lama per bisturi (taglia 15), un piccolo tampone, una pinzetta, un ago da 27 G, una sutura non riassorbibile 5-0, forbici e un porta-aghi nella finestra strumento microsurgical.</li>
	<li>Disimballare la vite intramidollare, il filo guida (0,3/0,2 mm di diametro, 10 cm di lunghezza), la punta di centraggio (0,5 mm di diametro) e il trapano a mano (Figura 1; Vedi Tabella materiali). Nota: La vite intramidollare (0,5 mm di diametro, 17,2 mm di lunghezza) è fatta in acciaio inox medicale per impianto retrogrado nel femore. La vite ha una filettatura prossimale (diametro 0,5 mm, 4 mm di lunghezza) con un naso (0,2 mm di diametro, lunghezza 0,4 mm) in punta e distale testa a forma di cono (diametro 0,8 mm, 0,9 mm di lunghezza) per raggiungere la frattura di compressione, nonché stabilità assiale e rotazionale.</li>
	<li>Esporre gli impianti e tutti gli strumenti chirurgici per una soluzione disinfettante (96% di alcool) per 5 min o sterilizzarli (sterilizzazione a vapore, 130 ° C, 25 min). Dopo la disinfezione o la sterilizzazione, mettere gli strumenti su un panno di operazione. Posizionare il panno di funzionamento direttamente adiacente alla tabella piccola operazione animale.</li>
</ol>2. gli animali, l'anestesia e Analgesia
<ol><li>Scegliere il ceppo, età e sesso dei topi secondo la domanda di studio che si rivolge. Nota: Per questo studio sono stati utilizzati topi CD-1 maschi di 12 a 14-week-old. Il peso del corpo appropriato utilizzare la vite di intramidollare è tra 25-35 g.</li>
	<li>Anestetizzare i topi con un'iniezione intraperitoneale di 15 mg/kg xylazina e 75 mg/kg ketamina. Confermare l'amputate pizzico di punta. Applicare il lubrificante di occhio per proteggere gli occhi degli animali da essiccazione durante l'anestesia. Dopo induzione dell'anestesia, posizionare il mouse sotto un radiatore di calore per mantenere costante la temperatura corporea. Durante la procedura, gli animali sono stati controllati con pizzico di ripetute punta a garantire un adeguato piano di anestesia.</li>
	<li>Applicare tramadol-cloridrato nell'acqua potabile (1,0 mg/mL) per analgesia dal 1 ° giorno prima dell'intervento chirurgico fino al 3 ° giorno dopo l'intervento chirurgico. Nota: Analgesia e infezione prevenzione dovrebbe essere d'accordo con le rispettive linee guida del paese e istituzione dove gli esperimenti devono essere eseguiti.</li>
</ol>3. intervento chirurgico e l'impianto a vite intramidollare
<ol><li>Prima dell'intervento chirurgico, radere tutta la gamba destra posteriore e applicare una crema depilatoria. Dopo 5 minuti, rimuovere la crema e pulire l'arto con acqua. Quindi, applicare una soluzione disinfettante con alcool al 96%. Clorexidina o Betadine può aggiungersi all'alcool per garantire completa asepsi.</li>
	<li>In condizioni asettiche, posizionare il mouse nella posizione supina sul tavolo piccola operazione animale. Piegare il ginocchio destro per consentire un approccio anteriore per i condili del ginocchio. Eseguire un'incisione di 5 mm pararotulea mediale al ginocchio destro utilizzando la lama del bisturi.</li>
	<li>Mobilitare il legamento patellar attentamente con il bisturi e il tampone. Quindi, spostare lateralmente la rotula con una pinzetta per esporre intercondiloidea del femore.</li>
	<li>Aprire la tacca intercondylar esattamente nel mezzo del femore tra entrambi i condili. Assicurarsi che non deve superare 1,0 mm di profondità per il foro di trapano.<ol>
<li>Avvio manuale di perforazione ad una velocità lenta e un offset 45 ° ventralmente all'asse del femore con 0,5 mm di centraggio trapano e il trapano a mano (Figura 1 e D, Figura 2). Durante la perforazione, continuamente diminuire l'angolo di offset di 0 ° (parallelo all'asse dell'osso del femore). Interrompere quando viene raggiunta una profondità di 1,0 mm di perforazione.</li>
	</ol>
</li>
	<li>Dopo l'apertura dell'osso alla tacca intercondylar, inserire l'ago 27 G nella cavità intramidollare su tutta la lunghezza del femore. Scremare la cavità intramidollare del femore manualmente attraverso movimenti rotativi dell'ago 27 G. Spingere l'ago avanti per perforare l'osso corticale alle grande trocantere prossimalmente.</li>
	<li>Rimuovere l'ago 27G e applicare il filo guida attraverso la parte distale del femore.<ol>
<li>Fare un'incisione della pelle con un bisturi (taglia 15) prossimalmente sopra il filo guida e spingere in avanti il filo guida fino a quando entrambe le estremità del filo guida sono fuori. Assicurarsi di mantenere il filo guida in posizione.</li>
	</ol>
</li>
	<li>Creare una frattura chiusa definita utilizzando la ghigliottina.<ol>
<li>Posizionare il mouse in posizione laterale con la gamba destra sotto la ghigliottina. Assicurarsi che la parte della diafisi del femore è posto nel mezzo della ghigliottina.</li>
		<li>Eliminare il peso (200 g) dalla distanza definita di 25,5 cm.</li>
	</ol>
</li>
	<li>Controllare la configurazione di frattura e frattura posizione così come la posizione del filo guida (Figura 3) utilizzando il dispositivo a raggi x (Vedi Tabella materiali).</li>
	<li>Collegare la vite intramidollare con il naso all'estremità distale per il filo di guida di 0.2 mm e inserirla nel femore sotto pressione continua e la rotazione in senso orario.<ol>
<li>Taglio dell'albero motore quando viene raggiunta la coppia sufficiente.</li>
		<li>Rimuovere il filo guida prossimalmente.</li>
	</ol>
</li>
	<li>Riposizionare la rotula e fissare il tendine della rotula ai muscoli con una sutura singola utilizzando un sintetico 5-0, monofilamento, nonabsorbable sutura in polipropilene. Utilizzare suture singole dello stesso materiale e dimensione per chiudere la ferita. Controllare la riduzione dei frammenti e la posizione della vite radiologicamente utilizzando il dispositivo a raggi x (Vedi Tabella materiali).</li>
	<li>Tenere gli animali sotto il radiatore di calore fino a quando recuperano dall'anestesia. Non lasciare gli animali incustoditi fino a quando essi hanno ripreso conoscenza sufficiente per mantenere decubito ventrale. Restituire gli animali nelle gabbie singole nella struttura degli animali. Non restituire gli animali per la compagnia di altri animali durante le prime 24 ore, anche se hanno pienamente recuperato dall'anestesia.</li>
	<li>Monitorare gli animali con cura ogni giorno. Mantenere l'analgesia postoperatoria usando tramadol cloridrato in acqua potabile con un dosaggio di 1,0 mg/mL durante i primi tre giorni. Continuare l'analgesia se, il giorno 4 dopo la chirurgia, gli animali mostrano ancora prova del dolore, come indicato dalla vocalizzazione, irrequietezza, mancanza di mobilità, il mancato sposo, postura anormale e mancanza di normale interesse nei dintorni. Terminare l'analgesia quando gli animali sono senza dolore.</li>
	<li>Alla fine dell'esperimento eutanasia animale da un'overdose di barbiturici.</li>
</ol>

<ol>
	<li>Jacenko, O., Olsen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mouse+models+in+studies+of+skeletal+disorders.">Transgenic mouse models in studies of skeletal disorders.</a> J Rheumatol Suppl. 43, 39-41 (1995).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+bone+healing+models:+standards,+tips,+and+pitfalls+results+of+a+consensus+meeting.">Small animal bone healing models: standards, tips, and pitfalls results of a consensus meeting.</a> Bone. 49 (4), 591-599 (2011).</li><li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal+bone.">Production of a standard closed fracture in laboratory animal bone.</a> J Orthop Res. 2 (1), 97-101 (1984).</li><li>Holstein, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+stable+closed+femoral+fracture+model+in+mice.">Development of a stable closed femoral fracture model in mice.</a> J Surg Res. 153 (1), 71-75 (2009).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+analysis+of+rotational+stiffness+of+different+osteosynthesis+techniques+in+mouse+femur+fracture.">Ex vivo analysis of rotational stiffness of different osteosynthesis techniques in mouse femur fracture.</a> J Orthop Res. 27 (9), 1152-1156 (2009).</li><li>Claes, L., Augat, P., Suger, G., Wilke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+size+and+stability+of+the+osteotomy+gap+on+the+success+of+fracture+healing.">Influence of size and stability of the osteotomy gap on the success of fracture healing.</a> J Orthop Res. 15 (4), 577-584 (1997).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+healing+process+in+non-stabilized+and+stabilized+femur+fractures+in+mice.">Characterization of the healing process in non-stabilized and stabilized femur fractures in mice.</a> Arch Orthop Trauma Surg. 136 (2), 203-211 (2016).</li><li>Thompson, Z., Miclau, T., Hu, D., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+intramembranous+ossification+during+fracture+healing.">A model for intramembranous ossification during fracture healing.</a> J Orthop Res. 20 (5), 1091-1098 (2002).</li><li>Cheung, K. M., Kaluarachi, K., Andrew, G., Lu, W., Chan, D., Cheah, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> J Orthop Res. 21 (4), 685-690 (2003).</li><li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+internal+fixation+of+femoral+fractures+in+mice:+impact+of+stability+on+fracture+healing.">A new technique for internal fixation of femoral fractures in mice: impact of stability on fracture healing.</a> J Biomech. 41 (8), 1689-1696 (2008).</li><li>Histing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model.">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> J Orthop Res. 28 (3), 397-402 (2010).</li><li>Garcia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LockingMouseNail-a+new+implant+for+standardized+stable+osteosynthesis+in+mice.">The LockingMouseNail-a new implant for standardized stable osteosynthesis in mice.</a> J Surg Res. 169 (2), 220-226 (2011).</li><li>Histing, T., Klein, M., Stieger, A., Stenger, D., Steck, R., Matthys, R., Holstein, J. H., Garcia, P., Pohlemann, T., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+to+analyze+metaphyseal+bone+healing+in+mice.">A new model to analyze metaphyseal bone healing in mice.</a> J Surg Res. 178 (2), 715-721 (2012).</li><li>Histing, T., Menger, M. D., Pohlemann, T., Matthys, R., Fritz, T., Garcia, P., Klein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Intramedullary+Locking+Nail+for+Standardized+Fixation+of+Femur+Osteotomies+to+Analyze+Normal+and+Defective+Bone+Healing+in+Mice.">An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice.</a> J Vis Exp. (117), (2016).</li><li>Hiltunen, A., Vuorio, E., Aro, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+experimental+fracture+in+the+mouse+tibia.">A standardized experimental fracture in the mouse tibia.</a> J Orthop Res. 11 (2), 305-312 (1993).</li><li>Manigrasso, M. B., O'Connor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+closed+femur+fracture+model+in+mice.">Characterization of a closed femur fracture model in mice.</a> J Orthop Trauma. 18 (10), 687-695 (2004).</li><li>Holstein, J. H., Menger, M. D., Culemann, U., Meier, C., Pohlemann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+locking+femur+nail+for+mice.">Development of a locking femur nail for mice.</a> J Biomech. 40 (1), 215-219 (2007).</li><li>Claes, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+mechanical+factors+on+the+fracture+healing+process.">Effects of mechanical factors on the fracture healing process.</a> Clin Orthop Relat Res. 355 Suppl, S132-S147 (1998).</li></ol>

Gli autori dichiarano di non avere nessun concorrenti interessi finanziari.

Fasi critiche della procedura chirurgica sono per trovare il punto di ingresso corretto per l'impianto di vite in mezzo i condili del femore a tacca intercondylar, nonché l'orientamento ottima dell'ago parallelo all'asse dell'osso per l'alesatura della cavità intramidollare. Per evitare una posizione di voce non corretta, il chirurgo dovrebbe preparare la tacca fino a quando si ottiene una vista ottimale. Per controllare l'orientamento durante la scrematura, femore dei topi deve essere tenuto con le dita in una posizio...

Guarigione ossea condotti in topi sono molto richiesti a causa di un ampio spettro di anticorpi e animali geneticamente modificati. Questi fatti permettono di studiare i meccanismi molecolari di1di guarigione ossea. Negli ultimi anni, diversa ossa guarigione modelli per topi sono stati sviluppati2. Questi modelli possono essere diviso in modelli aperti, in cui l'osso è osteotomized utilizzando un approccio chirurgico laterale aperto e in modelli chiusi, in cui l'osso è fratturato sulla base del modello di frattura introdotto da Bonnares ed Einhorn3. Utilizzando questa tecnica, una frattura trasversale standardizzata può essere prodotta da un dispositivo di piegatura di 3 punti e intramidollare impianti possono essere inseriti attraverso un'incisione pararotulea mediale piccolo in una tecnica mini-invasiva evitando un trauma dei tessuti molli principali.
La vite intramidollare può essere applicata per la stabilizzazione della frattura chiusa in topi. La vite offre stabilità rotazionale e assiale. Ciò si ottiene mediante compressione frattura attraverso un filo di prossimale e distale testa4. Ulteriori vantaggi della vite sono la semplice tecnica chirurgica, impiantare il basso grado di invasività, il basso peso e, soprattutto, una maggiore stabilità fornendo condizioni biomeccaniche standardizzate e controllate rispetto ad altri intramidollare gli impianti5. Infatti, nei modelli più chiusi di frattura, i frammenti sono stabilizzati solo da semplice PIN, che è associato con una completa mancanza di stabilità rotazionale e assiale e un elevato rischio di perno e anche frattura lussazione. Questo può influenzare notevolmente il processo di guarigione, che può provocare la guarigione ritardata o mancata unione formazione.
È noto che la stabilità della fissazione di frattura ha un enorme impatto sulla guarigione processo6,7. Una fissazione rigida alta risultati nella guarigione intramembranous, mentre una fissazione meno rigida, che potrebbe consentire micromovimenti del divario di frattura, con la conseguente guarigione endochondral. Stabilizzazione della frattura con la vite intramidollare Mostra principalmente un endochondral guarigione con una grande quantità di tessuto di callo, particolarmente dopo 2 settimane di guarigione di frattura. La possibilità di raccogliere una grande quantità di tessuto callo permette l'analisi di più parametri con tecniche diverse.
Qui, segnaliamo sulla progettazione e applicazione della vite intramidollare nei topi, così come sui suoi vantaggi e svantaggi in studi sperimentali su endochondral normale guarigione ossea.

<table><tbody><tr><td>Mouse Screw</td><td>RISystem AG</td><td>221,100</td><td></td></tr><tr><td>Guide wire</td><td>RISystem AG</td><td>521,100</td><td></td></tr><tr><td>Centering bit</td><td>RISystem AG</td><td>590,205</td><td></td></tr><tr><td>Hand drill</td><td>RISystem AG</td><td>390,130</td><td></td></tr><tr><td>Cotton-Swab (150 mm, small head)</td><td>Fink Walter GmbH</td><td>8822428</td><td></td></tr><tr><td>Suture (5-0 Prolene)</td><td>Ethicon</td><td>8614H</td><td></td></tr><tr><td>Forceps</td><td>Braun Aesculap AG &amp;amp;CoKG</td><td>BD520R</td><td></td></tr><tr><td>Scissors</td><td>Braun Aesculap AG &amp;amp;CoKG</td><td>BC100R</td><td></td></tr><tr><td>Needle holder</td><td>Braun Aesculap AG &amp;amp;CoKG</td><td>BM024R</td><td></td></tr><tr><td>27 G needle</td><td>Braun Melsungen AG</td><td>9186182</td><td></td></tr><tr><td>Scalpel blade size 15</td><td>Braun Aesculap AG &amp;amp;CoKG</td><td>16600525</td><td></td></tr><tr><td>Heat radiator</td><td>Sanitas</td><td>605.25</td><td></td></tr><tr><td>Depilatory cream</td><td>Asid bonz GmbH</td><td>NDXZ10</td><td></td></tr><tr><td>Eye lubricant</td><td>Bayer Vital GmbH</td><td>2182442</td><td></td></tr><tr><td>Xylazine</td><td>Bayer Vital GmbH</td><td>1320422</td><td></td></tr><tr><td>Ketamine</td><td>Serumwerke Bernburg</td><td>7005294</td><td></td></tr><tr><td>Tramadol</td><td>Gr&amp;uuml;nenthal GmbH</td><td>2256241</td><td></td></tr><tr><td>Disinfection solution (SoftaseptN)</td><td>Braun Melsungen AG</td><td>8505018</td><td></td></tr><tr><td>CD-1 mice</td><td>Charles River</td><td>22</td><td></td></tr><tr><td>X-ray Device</td><td>Faxitron MX-20, Faxitron X-ray Corporation</td><td>2321A0988</td><td></td></tr><tr><td>Fracture device small</td><td>RISystem AG</td><td>891,100</td><td></td></tr></tbody></table>

a minimally invasive model to analyze endochondral fracture healing in mice under standardized biomechanical conditions

Modelli di guarigione ossea sono necessari per analizzare i complessi meccanismi di frattura che guarisce per migliorare il trattamento delle fratture cliniche. Durante l'ultimo decennio, è stato notato un maggiore utilizzo di modelli murini in ricerca ortopedica, molto probabilmente perché i modelli murini offrono un gran numero di ceppi geneticamente modificati e particolari anticorpi per l'analisi dei meccanismi molecolari della guarigione della frattura. Per controllare le condizioni biomeccaniche, tecniche di osteosintesi ben caratterizzati sono obbligatorie, anche nei topi. Qui, segnaliamo sulla progettazione e l'uso di un osso chiuso modello curativo per stabilizzare le fratture di femore nei topi. La vite intramidollare, in acciaio inox medicale, fornisce attraverso compressione frattura una stabilità assiale e rotazionale rispetto ai perni intramidollari semplice usato principalmente, che mostrano una completa mancanza di stabilità assiale e rotazionale. La stabilità raggiunta dalla vite intramidollare permette l'analisi di endochondral guarigione. Una grande quantità di tessuto di callo, ricevuto dopo stabilizzazione con la vite, offre le condizioni ideali per la raccolta del tessuto per le analisi biochimiche e molecolari. Un ulteriore vantaggio dell'uso della vite è il fatto che la vite può essere inserita nel femore con tecnica mininvasiva senza indurre danni al tessuto molle. In conclusione, la vite è un unico impianto che può essere utilizzato idealmente in frattura chiusa guarigione modelli che offrono condizioni biomeccaniche standardizzate.

Il tempo di funzionamento da incisione cutanea alla chiusura della ferita era 20 min. La chirurgia può essere eseguita senza stereo-microscopio. Postoperatorio, gli animali sono stati controllati giornalmente. L'analgesia postoperatoria è stata terminata dopo 2 giorni perché nessuno degli animali ha mostrato la prova del dolore dopo questo periodo di tempo. Gli animali hanno mostrato anche normale peso-cuscinetto entro 2 giorni dopo la chirurgia. Le infezioni della ferita non sono stat...

Watch this Scientific Journal Video about A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions at JoVE.com

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

Questo protocollo descrive una tecnica di osteosintesi mininvasiva utilizzando una vite intramidollare per standardizzato stabilizzazione delle fratture di femore, che può essere usato per analizzare endocondrale ossa guarigione nei topi.

Un modello come minimo dilagante per analizzare Endochondral frattura la guarigione nei topi in condizioni standardizzate di biomeccaniche

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

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8.2K Views.  Saarland University. Questo protocollo descrive una tecnica di osteosintesi mininvasiva utilizzando una vite intramidollare per standardizzato stabilizzazione delle fratture di femore, che può essere usato per analizzare endocondrale ossa guarigione nei topi.

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

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.

Un semplice Critical dimensioni femorale Difetto Modello 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 ...

Ricerca

Medicina

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.

Istituzione di un segmentale femorale critico-size difetto del modello in topi stabilizzati da osteosintesi Piastra

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 ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Visualizzazione in tempo reale e analisi di condrociti lesioni dovute a carichi meccanici in espianti di cartilagine murina completamente intatta

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Bioingegneria

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

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

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

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

In Vivo Valutazione dello sviluppo di callo di frattura durante la guarigione dell'osso nei topi utilizzando un MRI-compatibile con osteosintesi per il femore di Mouse

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

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.

Un modello murino di chirurgia ortopedica per studiare la disfunzione cognitiva postoperatoria e rigenerazione tissutale

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

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.

La generazione delle fratture chiuse femorale in topi: un modello per studiare la guarigione ossea

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

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

Valutazione istomormetrica standardizzata dell'osteoartrite in un modello di topo chirurgico

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

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.

Frattura trasversale del femore del topo con perno stabilizzante

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 ...

Biologia

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.

Stabilire un modello di frattura del femore diafisario nei topi

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

<meta charset="utf8"></head><body>Protocollo standardizzato per la frattura mandibolare per la riparazione ossea nei topi

Less-Invasive Technique for Non-stabilized Mandibular Fracture in Mouse Models

Non-stabilized fractures can be made at mandibular sites in mice, thus making it possible to analyze bone repair using an endochondral ossification mode. To most accurately reflect this process in vivo, it is necessary to have a standardized protocol to avoid excessive bone loss and soft tissue damage, particularly at the mandibular site, an anatomical site characterized by minimal access. To our knowledge, we describe for the first time a less-invasive protocol of non-stabilized mandibular fracture in mice. Adult mice are anesthetized with isoflurane and receive a preoperative dose of buprenorphine by subcutaneous injection. A submandibular approach is performed, with a skin incision made along the inferior border of the mandible. The masseter muscle is elevated in a subperiosteal plane along the mandibular ramus with a periosteal elevator. A vertical and complete fracture of the ramus is performed from the basilar border to the coronoid notch (between condylar and coronoid processes), using a piezoelectric bone surgery device under saline serum irrigation at the basilar edge and scissors to complete the fracture of the mandibular ramus. The skin approach is closed by silk sutures. This detailed procedure for non-stabilized mandibular fractures offers an efficient procedure allowing minimal bone loss and soft tissue damage. This technique ensures successful and consistent results to accurately reflect the process of endochondral bone repair in mouse models.

<meta charset="utf8"></head><body>Tecnica meno invasiva per la frattura mandibolare non stabilizzata in modelli murini

This protocol presents a comprehensive and efficient method for analyzing mandibular bone repair. We describe a reproducible technique for non-stabilized mandibular fracture in a mouse model, allowing analysis of the process of endochondral bone repair with minimal tissue damage and bone loss.

Tecnica meno invasiva per la frattura mandibolare non stabilizzata in modelli murini

Non-stabilized fractures can be made at mandibular sites in mice, thus making it possible to analyze bone repair using an endochondral ossification mode. ...