Este trabalho foi financiado pela RISystem AG, Davos, Suíça.

Todos os procedimentos foram realizados de acordo com as diretrizes do National Institutes of Health para o uso de animais experimentais e seguiram as orientações institucionais (turismo für Verbraucherschutz, Zentralstelle Amtstierärztlicher Dienst, Saarbrücken Alemanha).
1. preparação de instrumentos cirúrgicos e implantes
<ol><li>Selecione uma lâmina de bisturi (tamanho 15), um pequeno cotonete, pinça fina, agulha 27G, uma sutura não absorvível de 5-0, tesoura e um porta-agulha da caixa instrumento microcirúrgico.</li>
	<li>Descompacte o parafuso da haste intramedular, o fio-guia (0,2/0,3 mm de diâmetro, 10 cm de comprimento), o bocado de broca de centragem (0,5 mm de diâmetro) e a broca de mão (Figura 1; consulte Tabela de materiais). Nota: O parafuso de haste intramedular (0,5 mm de diâmetro, comprimento de 17,2 milímetros) é feito de aço inoxidável de grau médico para implantação retrógrada em fêmur. O parafuso tem um segmento proximal (0,5 mm de diâmetro, comprimento de 4 mm) com um nariz (0,2 mm de diâmetro, 0,4 mm de comprimento) na ponta e distal em forma de cone de cabeça (0,8 mm de diâmetro, comprimento de 0,9 mm) para alcançar a fratura de compressão, bem como a estabilidade axial e rotacional.</li>
	<li>Expor os implantes e todos os instrumentos cirúrgicos para uma solução desinfectante (96% de álcool) por 5 min ou esterilizá-los (esterilização a vapor, 130 ° C, 25 min). Após a desinfecção ou esterilização, coloque os instrumentos sobre um pano de operação. O pano de operação diretamente adjacente à tabela pequena operação animais de posição.</li>
</ol>2. animais, anestesia e Analgesia
<ol><li>Escolha a tensão, idade e sexo dos ratos de acordo com a questão de estudo, que é dirigida. Nota: Para este estudo foram utilizados camundongos CD-1 sexo masculino de 12 a 14 semanas de idade. O peso de corpo adequado para usar o parafuso intramedular é entre 25-35 g.</li>
	<li>Anestesia os ratos com uma injeção intraperitoneal de 15 mg/kg de xilazina e 75 mg/kg de ketamina. Confirme o anesthetization pitada de dedo do pé. Aplique o lubrificante do olho para proteger os olhos dos animais de secagem durante a anestesia. Após a indução da anestesia, posicione o mouse sob um radiador de calor para manter a temperatura corporal constante. Durante o procedimento, os animais foram monitorados com pitada de repetidas do dedo do pé para garantir um plano adequado de anestesia.</li>
	<li>Aplica o cloridrato de tramadol em água potável (1,0 mg/mL) para analgesia dia 1, antes da cirurgia, até dia 3, após a cirurgia. Nota: Prevenção de infecção e Analgesia deve ser de acordo com as respectivas orientações do país e a instituição onde os experimentos devem ser executadas.</li>
</ol>3. ato cirúrgico e implantação do parafuso de haste intramedular
<ol><li>Antes da cirurgia, raspar toda a perna direita traseira e aplique um creme depilatório. Após 5 min, retire o creme e limpe a perna com água. Em seguida, aplica uma solução desinfectante com 96% de álcool. Betadine ou clorexidina pode ser adicionada para o álcool para garantir total assepsia.</li>
	<li>Sob condições assépticas, posicione o mouse na posição supina na mesa de operação de animais pequenos. Dobre o joelho direito para permitir uma abordagem anterior para os côndilos do joelho. Realize uma incisão parapatelar medial 5 mm no joelho direito usando a lâmina de bisturi.</li>
	<li>Mobilize o ligamento patelar cuidadosamente com a lâmina de bisturi e a amostra. Em seguida, deslocar a patela lateralmente com a pinça fina para expor o entalhe intercondilar do fêmur.</li>
	<li>Abra o entalhe intercondilar exatamente no meio do fêmur entre ambos os côndilos. Certifique-se de não deve exceder 1,0 mm de profundidade para o furo.<ol>
<li>Inicie-manual de perfuração em uma velocidade lenta e um deslocamento de 45 ° ventralmente ao eixo do fêmur com a 0,5 mm, centrando a broca e a broca de mão (Figura 1 e D, Figura 2). Durante a perfuração, continuamente diminua o ângulo de deslocamento de 0 ° (em paralelo com o eixo do osso do fêmur). Pare quando é alcançada uma profundidade de 1,0 mm de perfuração.</li>
	</ol>
</li>
	<li>Depois de abrir o osso que o entalhe intercondilar, introduza a agulha 27G na cavidade intramedular a todo o comprimento do fêmur. Resma de cavidade intramedular do fêmur manualmente através de movimentos giratórios da agulha 27G. Empurre a agulha para a frente para perfurar o osso cortical no grande trocanter proximalmente.</li>
	<li>Retire a agulha 27G e aplicar o fio-guia através da parte distal do fêmur.<ol>
<li>Fazer uma incisão na pele com uma lâmina de bisturi (tamanho 15) proximalmente sobre o fio guia e avance o fio-guia até que ambas as extremidades do fio-guia estão fora. Certifique-se de manter o fio-guia no lugar.</li>
	</ol>
</li>
	<li>Crie uma fratura fechada definida usando a guilhotina.<ol>
<li>Coloque o mouse em posição lateral com a perna direita na guilhotina. Certifique-se de que a parte de diáfise do fêmur é colocada no meio da guilhotina.</li>
		<li>Larga o peso (200 g) da distância definida de 25,5 cm.</li>
	</ol>
</li>
	<li>Controlar a configuração de fratura e fratura posição, bem como a posição do fio-guia (Figura 3), usando o aparelho de raio x (ver Tabela de materiais).</li>
	<li>Conecte o parafuso de haste intramedular com o nariz na extremidade distal para o fio-guia de 0.2 mm e inseri-lo no fêmur sob pressão contínua e a rotação no sentido horário.<ol>
<li>Cisalhamento do eixo rígido quando é alcançado o torque suficiente.</li>
		<li>Retire o fio-guia proximalmente.</li>
	</ol>
</li>
	<li>Reposicionar a patela e corrigir o tendão da patela aos músculos com uma sutura simples usando um sintético 5-0, monofilamento, sutura não absorvível de polipropileno. Use o única suturas do mesmo material e tamanho para fechar o ferimento. A redução dos fragmentos e a posição do parafuso radiologicamente usando o aparelho de raio-x de controle (veja a Tabela de materiais).</li>
	<li>Manter os animais sob o radiador de calor, até se recuperarem da anestesia. Não deixe os animais sem vigilância até eles recuperaram a consciência suficiente para manter a prostração ventral. Devolver os animais para gaiolas simples na instalação de animais. Não devolva os animais para a companhia de outros animais durante as primeiras 24 horas, mesmo que eles se recuperaram totalmente da anestesia.</li>
	<li>Monitore os animais com cuidado todos os dias. Manter a analgesia pós-operatória usando cloridrato de tramadol em água potável com uma dose de 1,0 mg/mL durante os três primeiros dias. Continue a analgesia se, no dia 4, após a cirurgia, os animais ainda mostram evidências de dor, conforme indicado pela vocalização, inquietude, falta de mobilidade, falha ao noivo, postura anormal e falta de interesse normal no ambiente. Finalizar a analgesia, quando os animais estão sem dor.</li>
	<li>No final do experimento abater o animal por uma overdose de barbitúricos.</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>

Os autores declaram que eles têm não tem interesses financeiro concorrente.

Passos críticos do procedimento cirúrgico são para encontrar o ponto de entrada correta para implantação do parafuso no meio os côndilos do fêmur no entalhe intercondilar, bem como a orientação ideal da agulha paralela ao eixo do osso para fresagem do cavidade intramedular. Para evitar uma posição de entrada incorreta, o cirurgião deve preparar o entalhe até que seja alcançada uma visão ideal. Para controlar a orientação durante a fresagem, o fêmur dos ratos deve ser realizado com os dedos em uma posiç...

Estudos de cura óssea em ratos estão em grande demanda por causa de um amplo espectro de anticorpos e animais geneticamente modificados. Estes factos permitem estudar os mecanismos moleculares do osso cura1. Nos últimos anos, osso de diferente modelos de cura para os ratos foram desenvolvidos2. Estes modelos podem ser divididos em modelos abertos, em que o osso é osteotomizado usando uma abordagem cirúrgica lateral aberta e em modelos fechados, baseados no modelo apresentado por Bonnares e Einhorn3fratura em que o osso está fraturado. Usando esta técnica, uma fractura transversal padronizada pode ser produzida por um dispositivo de dobrando de 3 pontos e implantes intramedulares podem ser inseridos através de uma incisão parapatelar medial pequeno em uma técnica minimamente invasiva, evitando um trauma de tecido mole principais.
O parafuso de haste intramedular pode ser aplicado para a estabilização da fratura fechada em camundongos. O parafuso oferece estabilidade rotacional e axial. Isto é conseguido por compressão de fratura através de um segmento proximal e uma distal cabeça4. Outras vantagens do parafuso são a técnica cirúrgica simples, o baixo grau de invasivity, o baixo peso e, principalmente, uma maior estabilidade, proporcionando condições biomecânicas padronizadas e controladas, em comparação com outro intramedular do implante implantes de5. Na verdade, nos modelos mais fechados da fratura, os fragmentos são estabilizados somente por pinos simples, que está associado uma completa falta de estabilidade rotacional e axial e um risco elevado de pin em também fratura luxação. Isto marcadamente pode influenciar o processo de cicatrização, que pode resultar em cicatrização ou formação não-sindicalizados.
É sabido que a estabilidade da fixação da fratura tem um impacto tremendo no processo cura6,7. Uma fixação rígida alta resulta em cura intramembranosa, enquanto uma fixação menos rígida, que pode permitir micromovimentos a lacuna de fratura, resultados na cura de endocondral. Estabilização da fratura com o parafuso de haste intramedular mostra predominantemente um endocondral cura com uma grande quantidade de calo de tecido, particularmente após duas semanas de consolidação da fratura. A possibilidade de colher uma grande quantidade de calo de tecido permite a análise de vários parâmetros por diferentes técnicas.
Aqui, nós relatamos sobre a concepção e a aplicação do parafuso intramedular em ratos, bem como sobre suas vantagens e desvantagens em estudos experimentais sobre a cura de osso endocondral normal.

<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

Modelos de cura óssea são necessários para analisar os mecanismos complexos de fratura cura para melhorar o tratamento clínico da fratura. Durante a última década, um aumento da utilização de modelos de rato em pesquisa ortopédica observou-se, muito provavelmente porque os modelos do rato oferecem um grande número de variedades geneticamente modificadas e anticorpos especiais para a análise dos mecanismos moleculares de consolidação da fratura. Para controlar as condições biomecânicas, técnicas de osteossíntese bem caracterizadas são obrigatórias, também em ratos. Aqui, nós relatamos no design e na utilização de uma modelo de consolidação fechada para estabilizar fraturas de fêmur em ratos. O parafuso da haste intramedular, feito de aço inoxidável de grau médico, fornece através da compressão da fratura uma estabilidade axial e rotacional, em comparação com os pinos intramedular simples mais utilizado, que mostram uma completa falta de estabilidade axial e rotacional. A estabilidade alcançada pelo parafuso intramedular permite a análise de endocondral cura. Uma grande quantidade de tecido de calo, recebido após a estabilização com o parafuso, oferece as condições ideais para a colheita de tecidos para análises bioquímicas e moleculares. Uma vantagem adicional do uso do parafuso é o fato de que o parafuso pode ser inserido em fêmur com uma técnica minimamente invasiva sem induzir danos no tecido macio. Em conclusão, o parafuso é um implante exclusivo que pode ser usado idealmente na fratura fechada modelos oferecendo condições biomecânicas padronizadas de cura.

O tempo de funcionamento da incisão de pele para fechamento de ferida foi de 20 min. A cirurgia pode ser realizada sem um estereoscópio. No pós-operatório, os animais foram monitorados diariamente. Analgesia pós-operatória foi finalizado depois de 2 dias, porque nenhum dos animais apresentou evidência de dor após este período de tempo. Os animais também mostraram peso-rolamento normal dentro de 2 dias após a cirurgia. Infecção da ferida não foram observadas durante o períod...

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

Este protocolo descreve uma técnica de osteossíntese minimamente invasiva usando um parafuso de haste intramedular padronizada estabilização de fraturas de fêmur, o que pode ser usado para analisar endocondral consolidação óssea em ratos.

Um modelo minimamente invasivo para analisar a fratura endocondral cura em ratos sob condições biomecânicas padronizadas

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

a-minimally-invasive-model-to-analyze-endochondral-fracture-healing

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.2K visualizações.  Saarland University. Este protocolo descreve uma técnica de osteossíntese minimamente invasiva usando um parafuso de haste intramedular padronizada estabilização de fraturas de fêmur, o que pode ser usado para analisar endocondral consolidação óssea em ratos.

Vídeo: 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.

Um Defeito modelo simples Critical porte Femoral em Ratos

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 ...

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.

Estabelecimento de um Defeito Modelo segmentar Femoral Critical-size em Ratos estabilizada por osteossíntese Placa

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.

Visualização em tempo real e análise do condrócito prejuízo devido ao carregamento mecânico em explantes de cartilagem murino totalmente intacta

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

Bioengenharia

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.

Na Vivo Avaliação do desenvolvimento de calo de fratura durante a consolidação óssea em ratos usando um dispositivo de osteossíntese compatível com MRI para o fêmur de 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.

Um modelo do rato de cirurgia ortopédica para estudar a disfunção cognitiva pós-operatória e regeneração de tecidos

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.

A geração de fraturas fechadas de fêmur em ratos: um modelo para estudar a consolidação óssea

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.

Avaliação histomorfométrica padronizada da osteoartrite em um modelo de rato cirúrgico

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.

Fratura transversa do fêmur do rato com pino estabilizador

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.

Estabelecimento de um Modelo de Fratura Diafisária do Fêmur em Camundongos

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>Protocolo padronizado de fratura mandibular para reparo ósseo em camundongos

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>Técnica menos invasiva para fratura mandibular não estabilizada em modelos de camundongos

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.

Técnica menos invasiva para fratura mandibular não estabilizada em modelos de camundongos

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