Name: the generation of closed femoral fractures in mice: a model to study bone healing
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Este trabajo fue financiado por donaciones desde el Departamento de defensa (DoD) nos ejército investigación médica y material comando (USAMRMC) Congreso dirigido médicos investigación programas (CDMRP) (PR121604) y los institutos Nacional de artritis y lesiones músculo-esqueléticas y piel (NIAMS), NIH R01 AR068332 a Uma Sankar.

El procedimiento se realizó con la aprobación de la Indiana University School de medicina institucional Animal Care y el Comité uso (IACUC). Todas las cirugías de supervivencia se realizaron bajo condiciones estériles como se indica en las directrices de los NIH. Dolor y el riesgo de infecciones fueron manejados con analgésicos apropiados y antibióticos para asegurar un resultado exitoso.
1. anestesia y preparación
<ol><li>Peso del ratón y anestesiar con una mezcla de ketamina (100 mg/kg) y xilacina (10 mg/kg) administrado a través de la ruta del intraperitoneal (I.P.). Coloque el ratón en una jaula vacía y vigilar hasta que está totalmente sedado.</li>
	<li>Asegúrese de que el ratón es sedado con un reflejo del pellizco del dedo del pie. Aplicar ungüento oftálmico en sus ojos para protegerlos de la desecación.</li>
	<li>Retire la piel de la extremidad derecha. Limpiar el sitio quirúrgico con un etanol de matorral y 70% base de yodo. Limpiar el sitio quirúrgico a partir del centro de la rodilla y haciendo un barrido circular hacia afuera. Repita este 3 x con peelings frescos, terminando con etanol al 70%.</li>
	<li>Administrar una dosis preoperatoria de analgesia clorhidrato de buprenorfina (0,03 mg/kg) por vía subcutánea para el tratamiento del dolor postoperatorio inmediato.</li>
	<li>Coloque el ratón sobre una almohadilla de calefacción cubierta por una esponja quirúrgica estéril.</li>
</ol>2. vía de abordaje
Nota: Antes de la fractura, el peso y la altura de caída deben empíricamente determinar la cepa específica, edad y sexo de los ratones antes de la cirugía. Este procedimiento quirúrgico está optimizado para ratones machos C57BJ6 en 10 semanas de edad.
<ol><li>Coloque el ratón en su parte posterior y flexión de la rodilla de la pierna operativa. Usando una hoja de bisturí, haga una incisión de 1.5 cm centrada sobre la articulación de la rodilla.</li>
	<li>Desplazar lateralmente la rótula utilizando pinzas para exponer el extremo distal del fémur. Inserte una aguja de hipodérmica 1.5 en acero inoxidable de largo calibre 25 en el centro de la ranura trochlear, a lo largo del canal medular en una manera retrógrada y a través del extremo proximal del fémur. Tomar una radiografía para asegurar la colocación correcta del perno. Nota: La aguja debe salir el lado dorsal del ratón para crear un camino para la guía.</li>
	<li>Pasar una guía de tungsteno larga 36 calibre 4 - in a través del eje de la aguja, entrando por el centro en el fémur distal y salir el bisel en el lado dorsal del ratón.</li>
	<li>Tras la exitosa colocación de la guía, retire con cuidado la aguja de calibre 25 tirando suavemente del cubo mientras sostiene la extremidad y la guía en su lugar. Confirmar la colocación de la guía por rayos x.</li>
	<li>Mantenga un peso de 391 g desde una altura de 34,6 cm por encima del disco de impacto (figura 1A). Apoyo a posición el fémur horizontal a través de los dos puntos, tal que las regiones intertrocantéricas y supracondíleo del fémur descansan sobre los yunques de apoyo (figura 1B) y el lado lateral de la extremidad hacia el punto de carga (figura 1 ). El peso de la gota y retire con cuidado el ratón desde el dispositivo inmediatamente después de la fractura.</li>
	<li>Confirmar la localización de la fractura mediante rayos x.</li>
	<li>Inserte la tubería hipodérmicas de acero inoxidable de calibre 24 sobre la guía metálica para estabilizar el fémur fracturado. Nota: Esta aplicación puede requerir una fuerza como el punto de entrada fue generado usando una aguja de un diámetro más pequeño. Esta diferencia de diámetro con eficacia previene una migración potencial de la varilla calibre 24 a través del extremo proximal del fémur. La profundidad de inserción se siente manualmente como el tubo de Romo encuentra la cortical ósea del trocánter mayor.</li>
	<li>Confirmar la posición de la barra de acero y la estabilización del fémur fracturado por rayos x antes de retirar la guía.</li>
	<li>Corte el exceso de tubo en el extremo distal del fémur con cortadores de alambre. Enterrar la tubería expuesta debajo de la superficie de los cóndilos con unas pinzas para aplicar una suave presión hacia abajo, teniendo cuidado de no dislocar la articulación de la rodilla.</li>
	<li>Vuelva a colocar la rótula usando fórceps. Cerrar la incisión con una sutura absorbible 5-0.</li>
</ol>3. cuidados posoperatorios
<ol><li>Después de la cirugía, los ratones se pueden inyectar con hasta 500 μl de estéril salina a través de la ruta I.P. para ayudar en su recuperación postoperatoria.</li>
	<li>Controlar los animales en una cama caliente de recuperación hasta que despierte de la cirugía. Una vez que ambulatorio, devolverlos a su jaula.</li>
	<li>Seguir vigilando de cerca los ratones durante varios días después de la cirugía para asegurarse de que están sanando correctamente y recuperar la movilidad. Administrar analgesia clorhidrato de buprenorfina (0,03 mg/kg) por vía subcutánea cada 6 h durante 3 días después de la cirugía y según sea necesario después de eso. Evitar el uso de antiinflamatorios no esteroideos (AINES), como se ha demostrado para deteriorar la cicatrización ósea después de la cirugía.</li>
</ol>

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Los autores de este manuscrito no tienen nada que revelar. El estado más de autores que no existen restricciones sobre el acceso completo a todo el material utilizado en el estudio divulgado en este manuscrito.

El objetivo de este procedimiento quirúrgico es generar fracturas femorales cerradas estandarizadas en ratones. Una ventaja clave de este modelo es que la fijación interna ocurre después de la generación de la fractura, evitando así una forma angular de la varilla intramedular. Quizás el aspecto más crítico de este protocolo es la generación de una fractura transversal estandarizada en el midshaft femoral, como la geometría de fractura depende de la fuerza de doblado aplicada y el posicionamiento de las extremi...

Fracturas son las lesiones más comunes que ocurren en el sistema músculo-esquelético y se asocian con una enorme carga socioeconómica, incluyendo los costos de tratamiento que se prevé para superar los $ 25 billones al año en los Estados Unidos1, 2. Aunque la mayoría de las fracturas cura sin incidente, curación se asocia con considerable tiempo de inactividad y pérdida de productividad. Aproximadamente 5-10% de todas las fracturas de resultar en un retraso cicatrización o no sindicalizados, debido a la edad u otras condiciones crónicas de salud subyacentes, tales como osteoporosis y diabetes mellitus3,4,5. No hay tratamientos farmacológicos aprobados por la FDA están disponibles actualmente para promover la curación ósea eficiente y acortar el tiempo de recuperación.
La curación de la fractura es un proceso complejo y altamente dinámico que involucra la coordinación de múltiples tipos de células. Por lo tanto, una comprensión global de los acontecimientos celulares y moleculares asociados con la regeneración ósea es crucial para la identificación de dianas terapéuticas que aceleran este proceso. Como con otras enfermedades humanas, el establecimiento de un modelo animal muy dócil y reproducible es crucial en el estudio de la curación del hueso. Animales más grandes, como ovejas y cerdos, propiedades de remodelación ósea y la biomecánica similar a los seres humanos, pero son caros, requieren mucho tiempo curativo y no son fácilmente susceptibles a manipulación genética6. Por otro lado, modelos animales pequeños, como ratas y ratones, ofrecen muchas ventajas, incluyendo una facilidad de manejo, bajo costo de mantenimiento, ciclos de reproducción cortos y una curación más corto tiempo7. Además, ratón secuenciado el genoma es completamente, lo que permite la rápida manipulación y generación de variantes genéticas. Así, el ratón es un sistema potente modelo para estudiar enfermedades humanas, lesiones y reparación de8. En los seres humanos, comorbilidades como diabetes mellitus y osteoporosis aumentan las posibilidades de una cicatrización retrasada. Varios de los actuales modelos de ratón están disponibles para estudiar los efectos de comorbilidades como diabetes mellitus y osteoporosis en lesiones de huesos y cicatrización. Pacientes que sufren de osteoporosis tienen una formación marcado disminuida del hueso durante las últimas etapas de una fractura cura9. Ratones (OVX) ovariectomizadas presentan pérdida ósea rápida y retrasada del hueso curativo similar a la observada en la osteoporosis postmenopáusica10,11. Además, muchos modelos de ratón de tipo I y tipo II diabetes imitan los fenotipos masa ósea baja y deterioro fractura cura visto en seres humanos11. Por otra parte, modelos murinos fractura sirven como una plataforma versátil para el estudio de los complejos procesos biológicos que ocurren en el callo y explorar nuevas estrategias terapéuticas que aceleran la regeneración del tejido óseo.
A pesar de las diferencias en la estructura ósea y el metabolismo, el proceso global de curación sigue siendo muy similar en ratones y seres humanos, la fractura de hueso que involucra una combinación de endocondral y Osificación intramembranosa seguido de remodelado óseo. La osificación endocondral involucra el reclutamiento de células progenitoras a regiones menos mecánicamente estables alrededor de la brecha de la fractura, donde se diferencian en condrocitos que hipertrofia y mineralizar el cartílago para producir un callo suave. La segunda ola de células progenitoras de infiltrarse en el callo y se diferencian en osteoblastos maduros que secretan nueva matriz de hueso12,13,de14,15. Durante la Osificación intramembranosa, progenitores en las superficies endóstica y periósticas directamente se diferencian en matriz secretando osteoblasts y facilitan los puentes de la fractura brecha9,11,12 ,13. Juntos, la osificaciones intramembranosa y la endocondral resultan en el desarrollo de un callo duro, que es remodelado más lejos en el tiempo para formar un hueso secundario fuerte capaz de soportar cargas mecánicas13,14 ,15. En los seres humanos sanos, el proceso de curación toma aproximadamente 3 meses, en comparación con sólo 35 días en ratones16.
Fracturas comúnmente se ha estudiado usando cualquiera de los modelos quirúrgicos abiertos o cerrados17. Abordajes quirúrgicos, tales como la generación de un defecto de tamaño crítico de abrir o completar la osteotomía, estandarizar la ubicación de la lesión y la geometría para reducir las desviaciones causadas por fracturas conminutas. Osteotomías sirven como un excelente modelo para estudiar el mecanismo subyacente detrás de una no unión debido a cicatrización se retrasa a menudo comparado con fracturas cerradas. Además, una fijación externa rígida es necesaria para estabilizar el hueso osteotomized, lo que significa que la regeneración dependerá principalmente de la Osificación intramembranosa. Abordajes quirúrgicos abiertos utilizan dispositivos tales como fijación de clavos, clavijas abrazaderas y placas de fijación para proporcionar estabilidad axial y rotacional de la extremidad fracturada; sin embargo, estos dispositivos son costosos y requieren mucho más tiempo en cirugía18,19,20,21. Por otro lado, los modelos cerrados se estabilizan con un dispositivo de fijación intramedular simple, que permite suficiente inestabilidad estimular la curación endocondral. Como resultado, modelos de fractura cerrada no imitar fácilmente las condiciones de una no Unión. Técnicas de fijación interna, como intramedular alfileres, clavos y tornillos de compresión, son ventajosas ya que son baratos, fácil de usar y minimizan el tiempo en cirugía21,,,2223. En algunos casos, pines intramedulares se insertan antes de la fractura, pero la flexión del pasador intramedular puede conducir a la angulación o desplazamiento del fémur fracturado, contribuyendo a un tamaño variable del callo y la curación. La localización de la fractura y la geometría son más difíciles de estandarizar en modelos cerrados, ya que se generan utilizando un dispositivo de flexión de tres puntos, en donde se deja caer un peso en la diáfisis. Sin embargo, con la técnica adecuada, esta vía de abordaje ofrece resultados rápidos y consistentes. Por otra parte, el modelo de fractura cerrada sirve como una herramienta clínicamente relevante para el estudio de las fracturas por impacto de alta fuerza o estrés mecánico22.
Este protocolo quirúrgico es una adaptación de los métodos anteriormente descritos utilizando un pin intramedular para estabilizar fracturas de fémur en ratas y ratones22,24,25. En primer lugar, se inserta una aguja intramedular de pequeño diámetro a través de la muesca intracondilar para establecer un punto de entrada, y se introduce una guía antes de generar una fractura transversal en el midshaft femoral usando un dependiente de la gravedad de tres puntos dispositivo de doblez. Tras la exitosa generación de una fractura cerrada de fémur, se incorpora una varilla intramedular de un diámetro más grande sobre el alambre guía para estabilizar el fémur fracturado. Este método evita el riesgo de cicatrización causada por la angulación de la pin intramedular durante la fractura, como la colocación de la fractura de la barra permite la estabilización optimizada y rotación del fémur lesionado.

<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

Fracturas óseas imponen una tremenda carga socio-económica en pacientes, además de afectar de manera significativa su calidad de vida. Estrategias terapéuticas que promueven la cicatrización ósea eficiente son inexistentes y en alta demanda. Modelos animales eficaces y reproducibles de curación de las fracturas son necesarios para entender los complejos procesos biológicos asociados a la regeneración del hueso. Muchos modelos animales de curación de fractura se han generado durante los años; sin embargo, modelos murinos fractura recientemente han surgido como poderosas herramientas para el estudio de cicatrización ósea. Se han desarrollado una variedad de modelos abiertos y cerrados, pero el modelo de fractura cerrada de fémur se caracteriza por ser un método sencillo para generar resultados rápidos y reproducibles de manera fisiológicamente relevante. El objetivo de este protocolo quirúrgico es generar fracturas femorales cerradas unilaterales en ratones y facilitar una estabilización de la fractura del fémur mediante la inserción de una varilla intramedular de acero. Aunque los dispositivos tales como un clavo o un tornillo ofrecen una mayor estabilidad axial y rotacional, el uso de una varilla intramedular proporciona una suficiente estabilización consistentes resultados curativos sin producir nuevos defectos en el tejido óseo o dañar cerca suave tejido. Proyección de imagen radiográfica se utiliza para supervisar la progresión de la formación de callo ósea Unión y posterior remodelación del callo óseo. Resultados curación ósea son típicamente asociados con la fuerza del hueso sanado y medidos con pruebas de torsión. Aún, comprender el tempranos eventos celulares y moleculares asociados con la reparación de la fractura es fundamental en el estudio de la regeneración del tejido óseo. El modelo de fractura cerrada de fémur en ratones con fijación intramedular sirve como una plataforma atractiva para estudiar hueso fractura cura y evaluar estrategias terapéuticas para acelerar la curación.

La exitosa implementación de la intervención quirúrgica fue supervisada con proyección de imagen radiográfica. Pasos clave incluyen la inserción de una aguja intramedular, la colocación de un alambre de guía, la inducción de una fractura transversal en el midshaft femoral y la estabilización adecuada con una varilla intramedular (figura 2A - 2Aiv). La progresión de la curación del callo de fractura fue supervisada...

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

El modelo murino fractura cerrada de fémur es una poderosa plataforma para el estudio de fracturas y nuevas estrategias terapéuticas para acelerar la regeneración ósea. El objetivo de este protocolo quirúrgico es generar fracturas femorales cerradas unilaterales en ratones con una varilla intramedular de acero para estabilizar el fémur.

La generación de fracturas femorales cerradas en ratones: un modelo para el estudio del hueso curativo

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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Universidad de Cartagena UDEC

Research

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JoVE Journal

Anatomy and Physiology

Medicine

Unit 1

Bone Tissue and the Skeletal System

SCIENTISTS IN ACTION

15.1K vistas.  Indiana University School of Medicine. El modelo murino fractura cerrada de fémur es una poderosa plataforma para el estudio de fracturas y nuevas estrategias terapéuticas para acelerar la regeneración ósea. El objetivo de este protocolo quirúrgico es generar fracturas femorales cerradas unilaterales en ratones con una varilla intramedular de acero para estabilizar el fémur.

Video: 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: Un Modelo periférica aguda trauma tisular

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

Investigación

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.

A-crítico tamaño femoral Defecto sencillo modelo en ratones

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.

Establecimiento de un defecto segmentario Modelo femoral de tamaño crítico en ratones estabilizado por osteosíntesis Plate

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.

Un clavo intramedular de bloqueo para estandarizada fijación del fémur osteotomías para analizar normal y defectuoso Bone Healing en ratones

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

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.

En Vivo Evaluación del desarrollo del callo de fractura durante la cicatrización ósea en ratones usando un dispositivo de osteosíntesis de MRI-compatible para el fémur de ratón

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 modelo de ratón de cirugía ortopédica para el estudio de la disfunción cognitiva postoperatoria y regeneración de los tejidos

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.

Un modelo mínimamente invasivo para analizar la fractura endocondral en ratones en condiciones biomecánicas estandarizadas

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

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 el desarrollo de un modelo de osteotomía de fémur en ratas albinas 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.

Fractura transversal del fémur del ratón con pasador 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 ...

Biología

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.

Establecimiento de un modelo de fractura de fémur diafisario en ratones

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