Este trabalho foi apoiado por concessões do departamento de defesa (DoD) nos exército pesquisa médica e Materiel comando (USAMRMC) Congressional dirigido Medical Research programas (CDMRP) (PR121604) e os institutos nacionais de artrite e osteomuscular e dermatoses (NIAMS), NIH R01 AR068332 para Uma Sankar.

O procedimento a seguir foi realizado com a aprovação do Comitê de uso (IACUC) e Indiana universidade escola de medicina institucional Cuidado Animal. Todas as cirurgias de sobrevivência foram realizadas sob condições estéreis, conforme descrito pelas diretrizes do NIH. Dor e risco de infecções eram geridas com adequada analgésicos e antibióticos para garantir um resultado positivo.
1. anestesia e preparação
<ol><li>Pesar o mouse e anestesiá-lo com uma mistura de cetamina (100 mg/kg) e xilazina (10mg/kg) administrada através de via intraperitoneal (I.P.). Coloque o mouse em uma gaiola vazia e monitorá-lo até que está totalmente sedado.</li>
	<li>Certifique-se de que o mouse está sedado usando um reflexo de pitada de dedo do pé. Aplica a pomada oftálmica para seus olhos para protegê-los da secagem.</li>
	<li>Retire a pele da extremidade direita. Limpe o local cirúrgico com um etanol de esfoliação e 70% à base de iodo. Esfregue o local cirúrgico, começando no centro do joelho e fazer uma varredura circular para fora. Repita este 3x com scrubs frescos, terminando com etanol a 70%.</li>
	<li>Administre uma dose pré-operatória de analgesia de cloridrato de buprenorfina (0,03 mg/kg) por via subcutânea para a gestão da dor no pós-operatório imediato.</li>
	<li>Coloque o mouse sobre uma almofada de aquecimento, coberta por um bloco cirúrgico estéril.</li>
</ol>2. abordagem cirúrgica
Nota: Antes da fratura, o peso e a altura de queda devem ser empiricamente determinadas para a cepa específica, idade e sexo dos ratos antes da cirurgia. Este procedimento cirúrgico é otimizado para camundongos machos C57BJ6 com 10 semanas de idade.
<ol><li>Coloque o mouse sobre as costas e flexionar o joelho da perna operativa. Usando uma lâmina de bisturi, faça uma incisão de 1,5 cm, centralizada sobre a articulação do joelho.</li>
	<li>Deslocar lateralmente à patela usando fórceps para expor a extremidade distal do fêmur. Inserir a agulha hipodérmica 1.5 - em aço inoxidável de longa 25 gauge no centro do sulco troclear, ao longo do canal medular de forma retrógrada e através da extremidade proximal do fêmur. Tire um raio-x para garantir a colocação apropriada do pino. Nota: A agulha deve sair do lado dorsal do mouse para criar um caminho para o fio-guia.</li>
	<li>Passe um fio-guia do tungstênio calibre 36 tempo 4 - na através do eixo da agulha, entrando através do hub no fêmur distal e saindo o chanfro do lado dorsal do mouse.</li>
	<li>Após a colocação bem sucedida do fio-guia, remova cuidadosamente a agulha de calibre 25 puxando suavemente no hub, mantendo o membro e o fio-guia no lugar. Confirme a colocação do fio-guia por raios-x.</li>
	<li>Segure um peso de 391 g de uma altura de 34,6 cm acima do disco de impacto (figura 1A). Posição do fêmur horizontalmente através de dois suportar pontos, tal que as regiões intertrocantérica e supracondilianas do fêmur descansar sobre os batentes de apoio (figura 1B) e o lado lateral do membro é virado para o ponto de carregamento (Figura 1 ). Largar o peso e Retire cuidadosamente o mouse do dispositivo imediatamente após a fratura.</li>
	<li>Confirme o local da fratura por raios-x.</li>
	<li>Insira o tubo de aço inoxidável de calibre 24 hipodérmico sobre o guia para estabilizar o fêmur fraturado. Nota: Este aplicativo pode exigir alguma força como o ponto de entrada foi gerado usando uma agulha de diâmetro menor. Esta diferença de diâmetro efetivamente impede uma migração potencial da haste calibre 24 através da extremidade proximal do fêmur. A profundidade de inserção pode ser sentida manualmente como a tubagem romba encontra o osso cortical do grande trocanter.</li>
	<li>Confirme a posição da haste de aço e a estabilização do fêmur fraturado por raio-x antes de remover o fio-guia.</li>
	<li>Corte o tubo em excesso na extremidade distal do fêmur usando cortadores de fio. Enterre os tubos expostos sob a superfície dos côndilos usando fórceps para aplicar uma força descendente suave, tomando cuidado para não deslocar a articulação do joelho.</li>
	<li>Reposicione a patela usando fórceps. Feche o local da incisão com uma sutura absorvível de 5-0.</li>
</ol>3. pós-operatórios
<ol><li>Após a cirurgia, os ratos podem ser injetados com até 500 µ l de estéril salina através da rota I.P. para ajuda-los na sua recuperação pós-operatória.</li>
	<li>Monitore os animais em uma cama de recuperação aquecida até que eles despertam da cirurgia. Uma vez ambulatorial, devolvê-los à sua jaula.</li>
	<li>Continue a acompanhar de perto os ratos durante vários dias após a cirurgia para garantir que eles são a cura adequadamente e recuperar a mobilidade. Administre analgesia de cloridrato de buprenorfina (0,03 mg/kg) por via subcutânea cada 6 h durante 3 dias após a cirurgia e conforme necessário depois disso. Evite o uso de drogas antiinflamatórias não-esteroides (AINEs), como eles têm sido mostrados para prejudicar a consolidação óssea após a cirurgia.</li>
</ol>

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Os autores deste manuscrito não tem nada para divulgar. Os autores ainda afirmam que não há restrições sobre o completo de todo o material utilizado no estudo relatado neste manuscrito.

O objetivo deste procedimento cirúrgico é gerar fraturas femorais fechadas padronizadas em camundongos. A principal vantagem desse modelo é que a fixação interna ocorre após a geração da fratura, evitando assim uma angulação da haste intramedular. Talvez o aspecto mais importante deste protocolo é a geração de uma fratura transversal padronizada no midshaft fêmur, como a geometria da fratura é dependente da força de flexão aplicada e o posicionamento do membro hind. Posicionamento incorreto do fêmur dur...

Fraturas estão entre as lesões mais comuns que ocorrem para o sistema músculo-esquelético e estão associadas com um enorme fardo socioeconômico, incluindo custos de tratamento que são projetados para superar a US $ 25 bilhões anualmente no Estados Unidos1, 2. Embora a maioria das fraturas cura sem incidentes, a cura é associado com substancial tempo de inatividade e perda de produtividade. Aproximadamente 5-10% de todas as fraturas resultar em um atraso de cura ou não-sindicalizados, devido à idade ou outras condições de saúde crônica subjacente, tais como osteoporose e diabetes mellitus3,4,5. Não há tratamentos farmacológicos aprovados pela FDA estão atualmente disponíveis para promover a cicatrização óssea eficiente e encurtar o tempo de recuperação.
Consolidação da fratura é um processo complexo e altamente dinâmico envolvendo a coordenação de vários tipos de células. Portanto, uma compreensão abrangente dos eventos celulares e moleculares associados a regeneração óssea é crucial para a identificação de alvos terapêuticos que acelerar este processo. Como com outras doenças humanas, o estabelecimento de um modelo animal altamente favorável e reprodutível é crucial no estudo da consolidação óssea. Animais maiores, como ovelhas e suínos, têm propriedades de remodelação óssea e biomecânica semelhantes aos seres humanos, mas são caros, exigem tempo cura substancial e não são facilmente passíveis de manipulação genética6. Por outro lado, os modelos animais pequenos, tais como ratos e camundongos, oferecem muitas vantagens, incluindo uma facilidade de manuseio, baixo custo de manutenção, ciclos de reprodução curto e um mais curto de tempo cura7. Além disso, o genoma do rato é totalmente sequenciado, permitindo a manipulação rápida e a geração de variantes genéticas. Assim, o mouse é um sistema poderoso modelo para estudar doenças humanas, lesão e reparar a8. Em humanos, comorbidades como osteoporose e diabetes mellitus aumentam a probabilidade de uma cicatrização. Um número de modelos existentes do mouse está disponíveis para estudar os efeitos de comorbidades, tais como osteoporose e diabetes mellitus na lesão do osso e cura. Pacientes que sofrem de osteoporose tem uma formação óssea diminuiu acentuadamente durante as fases posteriores de uma fratura cura9. Ovariectomizadas ratos (OVX) exibem perda óssea rápida e óssea retardada de cura semelhantes às observadas em osteoporose pós-menopausa10,11. Além disso, muitos modelos de mouse do tipo I e tipo diabetes II imitar a fenótipos massa óssea baixa e prejudicada consolidação visto em humanos11. Além disso, modelos de fratura murino servem como uma plataforma versátil para estudar os processos biológicos complexos ocorrem em calo ósseo e explorar novas estratégias terapêuticas que aceleram a regeneração do tecido ósseo.
Apesar das diferenças na estrutura óssea e metabolismo, o processo geral de fratura óssea cura permanece muito semelhante em camundongos e humanos, envolvendo uma combinação de endocondral e ossificação intramembranosa seguido de remodelação óssea. Ossificação endocondral envolve o recrutamento de células progenitoras para regiões menos mecanicamente estáveis em torno da gap de fratura, onde eles se diferenciar em condrócitos que hipertrofia e mineralize da cartilagem para produzir um calo mole. A segunda onda de células progenitoras infiltrar o calo e se diferenciam em osteoblastos maduros que secretam novo osso matriz12,13,14,15. Durante a ossificação intramembranosa, progenitores na superfície periosteal e endosteal diretamente diferenciarem em matriz secretoras de osteoblastos e facilitam a ponte da fratura lacuna9,11,12 ,13. Juntos, a endocondral e intramembranosa ossificações resultam no desenvolvimento de um calo duro, que é ainda mais remodelado ao longo do tempo para formar um osso secundário forte capaz de suportar cargas mecânicas13,14 ,15. Em seres humanos saudáveis, o processo de cicatrização leva cerca de 3 meses, em comparação com apenas 35 dias em ratos16.
Consolidação da fratura foi comumente estudado usando qualquer modelos cirúrgicos abertos ou fechados,17. Abra as abordagens cirúrgicas, como a geração de um defeito de tamanho crítico ou completar a osteotomia, padronizar a localização da lesão e a geometria para reduzir os desvios causados por fraturas cominutivas. Osteotomias servem como um excelente modelo para estudar o mecanismo subjacente por trás de um não-União porque a cura é frequentemente atrasada em comparação com fraturas fechadas. Além disso, uma fixação rígida externa é necessária para estabilizar o osso osteotomized, significando que a regeneração dependerá principalmente da ossificação intramembranosa. Abordagens cirúrgicas abertas usam dispositivos tais como bloqueio de unhas, pin-clipes e placas de travamento para fornecer estabilidade axial e rotacional ao membro fraturado; no entanto, tais dispositivos são caros e requerem significativamente mais tempo na cirurgia18,19,20,21. Por outro lado, modelos fechados são estabilizados com um dispositivo de fixação intramedular simples, permitindo a suficiente instabilidade estimular a cura endocondral. Como resultado, modelos de fratura fechada não facilmente imitar as condições de um não-sindicalizados. Técnicas de fixação interna, tais como pinos intramedular, pregos e parafusos de compressão, são vantajosas, como eles são mais barato, fácil de usar e minimizar o tempo de cirurgia21,22,23. Em alguns casos, os pinos intramedulares são inseridos antes da fratura, mas da flexão do pino intramedular pode levar a angulação ou deslocamento do fêmur fraturado, contribuindo para um tamanho variável calo e cura. O local da fratura e geometria são mais difíceis de padronizar em modelos fechados, como eles são gerados usando um dispositivo de flexão de três pontos, onde um peso é cair na diáfise. No entanto, com a técnica adequada, esta abordagem cirúrgica oferece resultados rápidos e consistentes. Além disso, o modelo de fratura fechada serve como uma ferramenta clinicamente relevante para o estudo de fraturas causadas por força de alto impacto ou estresse mecânico22.
Este protocolo cirúrgico foi adaptado de métodos anteriormente descritos usando um pino intramedular para estabilizar a fraturadas fêmures de ratos e camundongos22,24,25. Primeiro, uma agulha de haste intramedular de pequeno diâmetro é inserida através do entalhe de intracondylar para estabelecer um ponto de entrada, e um fio-guia é introduzido antes de gerar uma fractura transversal a femoral midshaft usando um três-ponto de gravidade-dependente dispositivo de dobra. Após a bem sucedida geração de uma fratura do fêmur fechada, uma haste intramedular de maior diâmetro é incorporada sobre o fio-guia para estabilizar o fêmur fraturado. Este método evita o risco de cicatrização causada pela angulação do pino intramedular durante a fratura, como a colocação da pós-fratura haste permite a estabilização de reposicionamento e otimizada do fêmur ferido.

<table><tbody><tr><td>Oster Minimax Trimmer</td><td>Animal World Network</td><td>78049-100</td><td></td></tr><tr><td>POVIDONE-IODINE</td><td>Thermo Fisher Scientific</td><td>395516</td><td></td></tr><tr><td>OPHTHALMIC OINTMENT</td><td>Thermo Fisher Scientific</td><td>NC0490117</td><td></td></tr><tr><td>Styker T/Pump Warm Water Recirculator</td><td>Kent Scientific Corporation</td><td>TP-700</td><td></td></tr><tr><td>1ml Sub-Q Syringe</td><td>Thermo Fisher Scientific</td><td>309597</td><td></td></tr><tr><td>ENCORE Sensi-Touch PF</td><td>Moore Medical LLC</td><td>30347</td><td>Latex, powder-free surgical glove</td></tr><tr><td>PrecisionGlide 25G Hypodermic Needles</td><td>Thermo Fisher Scientific</td><td>14-826-49</td><td></td></tr><tr><td>Ultra-High-Temperature Tungsten Wire,</td><td>McMaster-Carr</td><td>3775K37</td><td>0.005&quot; Diameter, 1/16 lb. Spool, 380&#039; Long</td></tr><tr><td>304 stainless steel, 24G thin walled tubing</td><td>Microgroup Inc</td><td>304h24tw-5ft</td><td></td></tr><tr><td>#15 Scalpel Blades</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>#10 Scalpel Blades</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>Narrow Pattern Forceps</td><td>Fine Science Tools</td><td>11002-12</td><td>Serrated/Straight/12cm</td></tr><tr><td>Iris Forceps</td><td>Fine Science Tools</td><td>11066-07</td><td>1x2 Teeth/Straight/7cm</td></tr><tr><td>Dissector Scissors</td><td>Fine Science Tools</td><td>14081-09</td><td>Slim Blades/Angled to Side/Sharp-Sharp/10cm</td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td>ToughCut/Straight/Sharp-Sharp/11.5cm</td></tr><tr><td>Olsen-Hegar Needle Holder with Suture Cutter</td><td>Fine Science Tools</td><td>12002-12</td><td>Straight/Serrated/12cm/with Lock</td></tr><tr><td>Crile Hemostat</td><td>Fine Science Tools</td><td>13004-14</td><td>Serrated/Straight/14cm</td></tr><tr><td>Tungsten Wire Cutter</td><td>ACE Surgical Supply Co., Inc.</td><td>08-051-90</td><td>ACE #150 Wire Cutter, tungsten carbide tips</td></tr><tr><td>3-0 VICRYL Suture</td><td>Ethicon Suture</td><td>J423H</td><td>3-0 VICRYL UNDYED 27&quot; FS-2 CUTTING</td></tr><tr><td>piXarray 100 Digital Specimen Radiography System</td><td>Bioptics, Inc</td><td></td><td>Cabinet x-ray system</td></tr><tr><td>Einhorn 3-Point Bending Device</td><td>N/A</td><td>N/A</td><td>Custom Built</td></tr></tbody></table>

the generation of closed femoral fractures in mice: a model to study bone healing

Fraturas ósseas imponham um enorme fardo socio-económica em pacientes, além de afetar significativamente a sua qualidade de vida. Estratégias terapêuticas que promovem a consolidação óssea eficiente são inexistentes e em alta demanda. Eficazes e reprodutíveis de modelos animais de cura de fraturas são necessários para compreender os complexos processos biológicos associados a regeneração óssea. Muitos modelos animais de consolidação da fratura foram gerados ao longo dos anos; no entanto, modelos de fratura murino surgiram recentemente como poderosas ferramentas para estudar a consolidação óssea. Desenvolveram-se uma variedade de modelos abertos e fechados, mas o modelo de fratura fechada do fêmur se destaca como um método simples para a geração de resultados rápidos e podem ser reproduzidos de forma fisiologicamente relevante. O objetivo do presente protocolo cirúrgico é gerar fraturas femorais fechadas unilaterais em camundongos e facilitar uma estabilização pós-fratura do fêmur através da inserção de uma haste intramedular aço. Apesar de dispositivos como um prego ou um parafuso para oferecer maior estabilidade axial e rotacional, o uso de uma haste intramedular fornece uma estabilização suficiente para resultados consistentes de curativas sem produzir novos defeitos no tecido ósseo ou danificar-se nas proximidades de macio tecido. Imagem radiográfica é usada para monitorar a progressão da formação de calo, união óssea e posterior remodelação do calo ósseo. Resultados de cicatrização óssea são normalmente associados com a força do osso curado e medidos com testes de torção. Ainda assim, compreendendo os início eventos celulares e moleculares associados a reparação da fratura é fundamental no estudo da regeneração do tecido ósseo. O modelo de fratura fechada de fêmur em ratos com fixação intramedular serve como uma plataforma atrativa para estudar a consolidação de fratura óssea e avaliar estratégias terapêuticas para acelerar a cicatrização.

O sucesso da implementação do procedimento cirúrgico foi monitorado com imagens radiográficas. Etapas-chave incluem a inserção de uma agulha de haste intramedular, a colocação de um fio-guia, a indução de uma fratura transversal no midshaft a femoral e a estabilização adequada com uma haste intramedular (Figura 2Aeu - 2Aiv). A progressão de cura do calo da fratura foi monitorada com imagens radiográficas semanai...

Watch this Scientific Journal Video about The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing at JoVE.com

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

O modelo murino fratura fechada do fêmur é uma plataforma poderosa para estudar a consolidação da fratura e novas estratégias terapêuticas para acelerar a regeneração óssea. O objetivo do presente protocolo cirúrgico é gerar fraturas femorais fechadas unilaterais em camundongos, usando uma haste intramedular aço para estabilizar o fêmur.

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 ...

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Anatomy and Physiology

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Unit 1

Bone Tissue and the Skeletal System

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15.1K visualizações.  Indiana University School of Medicine. O modelo murino fratura fechada do fêmur é uma plataforma poderosa para estudar a consolidação da fratura e novas estratégias terapêuticas para acelerar a regeneração óssea. O objetivo do presente protocolo cirúrgico é gerar fraturas femorais fechadas unilaterais em camundongos, usando uma haste intramedular aço para estabilizar o fêmur.

Vídeo: The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Pseudofracture: An Acute Peripheral Tissue Trauma Model

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

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

Pseudofracture: Um Modelo de trauma agudo do tecido periférico

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

Medicina

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 ...

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 ...

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.

Uma haste intramedular de bloqueio para Padronizado Fixação do fêmur Osteotomias para analisar normal e defeituosa cicatrização óssea em ratos

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

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 ...

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.

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 ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

Fratura aparelhos Design e otimização de protocolo para fraturas fechadas-estabilizado em roedores

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

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.

Protocolo para o Desenvolvimento de um Modelo de Osteotomia do Fêmur em Ratos Albinos Wistar

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.

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 ...