Nos gustaría dar las gracias al Dr. Commons George y el Dr. Dean Vistnes por su apoyo y colaboración de nuestra investigación. Este trabajo es apoyado por el Instituto Nacional de las subvenciones Investigación Dental y Craneofacial 1 DE019274 R21-01, R01EB009689 y DE020771 RC2-02, la Fundación Oak y Laboratorio de Medicina Pediátrica Hagey Regenerativa con MTL Dr. Hyun es apoyado por el Saint Joseph Mercy Hospital GME .

1. Celda de aislamiento y Expansión <ol><li> Todo el consentimiento del paciente y de los protocolos experimentales fueron revisados ​​y aprobados por la Universidad de Stanford Institutional Review Board (Protocolo # 2188 y # 9999). </li><li> Obtención de tejido adiposo subcutáneo humano de los procedimientos electivos bajo lipoaspiración local / anestesia general. </li><li> Habrá dos capas en el lipoaspirado (Figura 1A). El sobrenadante contiene la gran mayoría del material celular procesada. La capa inferior se inyecta solución salina en su mayoría. Adiposo derivado de células estromales se pueden cosechar a partir de cualquiera de las capas, pero el rendimiento es mucho mayor a partir del sobrenadante. </li><li> Para aislar la fracción del estroma vascular (SVF), lavar el lipoaspirado extensamente con volúmenes iguales de 1X tampón fosfato salino (PBS) que contenía 2,5% betadine (x2), seguido por un volumen igual de PBS 1X (x1) sin betadine. Permitir que el lavado a precipitar. </li><li> Tenga cuidado de mantener estéril technique durante todo el proceso ya que la contaminación con el tejido humano procesada puede suceder. </li><li> Aspirar la capa inferior y desechar después de cada lavado. </li><li> Alícuota de 15 ml de tejido adiposo en 50 ml tubos cónicos BD Falcon. </li><li> Se digiere el tejido adiposo con un volumen igual (15 ml) de filtrado 0,075% de colagenasa tipo II en solución salina equilibrada de Hank (HBSS) a 37 ° C en un baño de agua durante 60 min con agitación constante (aprox. 180 sacudidas / min) - ventilar cada tubo cada 15 min. </li><li> Neutralizar la actividad de la enzima con 15 ml de PBS que contenía 10% (suero bovino fetal) FBS, y se centrifuga a 1.000 rpm durante 5 min a 4 ° C para obtener una alta densidad SVF pellet. El sedimento será una mezcla de adiposas derivadas de las células del estroma y eritrocitos. </li><li> Descartar el sobrenadante sin perturbar el sedimento. </li><li> Resuspender el precipitado en 10 ml de medio de crecimiento tradicionales (Eagle modificado de Dulbecco Medium [DMEM] / 10% FBS / 1% de penicilina-estreptomicina solución). </li><li> Filtro de suspensión<html> sión a través de un filtro de células de nylon 100 micras para eliminar los restos celulares. </li><li> Combina 3 tubos a uno limpio cónico de 50 ml. Centrifugar a 1.000 rpm durante 5 min a 4 ° C. </li><li> Descartar el sobrenadante sin perturbar el sedimento. </li><li> Resuspender el sedimento en 5 ml de medio de crecimiento tradicionales </li><li> Combina dos cónicas 50 ml por placa de 10 cm o cinco cónicas 50 ml para una placa de 15 cm. </li><li> Establecer cultivos primarios durante la noche a 37 ° C/21% de O 2, 5% de CO 2. </li><li> Después de la incubación durante la noche, se lavan las placas exhaustivamente con PBS para retirar los residuos de las células no adherentes de sangre rojas. Las poblaciones celulares resultantes son adiposas derivadas de las células del estroma. </li><li> Mantener las células a niveles sub-confluentes para prevenir la diferenciación espontánea a 37 ° C/21% de O 2, 5% de CO 2 en un medio de crecimiento. Las células generalmente se deben dividir cada tres a cuatro días en medios de crecimiento regulares cuando se siembran al 50% de confluencia. </li></ol>ve_title &quot;&gt; 2. Preparación Andamio <ol><li> Los andamios fueron realizadas por el Dr. Min Lee en la Universidad de California, Los Angeles - Escuela de Odontología. </li><li> Andamios de PLGA fueron fabricados de 85/15 poli (láctico-co-glicólico) (viscosidad inherente = 0,61 dl / g, Birmingham Polymers) por moldeo con disolvente y un proceso de lavado de partículas. </li><li> PLGA / cloroformo soluciones se mezclaron con sacarosa 200-300 m de diámetro para obtener 92% de porosidad (fracción de volumen), y se comprimen para formar láminas delgadas en un molde de teflón. </li><li> Después de la liofilización durante la noche, andamios se sumergieron en tres cambios de doble destilada (dd) H 2 O para disolver la sacarosa, y suavemente retiró de la placa de teflón con una espátula de punta fina. </li><li> Después de lavado de partículas, todos los andamios se desinfectaron por inmersión en 50%, 60% y 70% de etanol durante 30 min cada uno, seguido de tres lavados de ddH 2 O. </li><li> Todos los andamios se secaron bajo una campana de flujo laminar. </li><li> Después de Fabricación de la estructura, scaffolds fueron recubiertos con apatita. <ol class="ualpha"><li> SBF (fluido corporal simulado) Se preparó una solución con concentraciones de iones que eran 5 veces la del plasma sanguíneo humano. Todas las soluciones son estériles filtró a través de una membrana de 0,22 micras PES (Nalgene). Inmediatamente antes de que el proceso de recubrimiento, se secó andamios de PLGA se someten a descarga de resplandor, argón-plasma de grabado (Harrick Scientific) para mejorar la humectación y la uniformidad del recubrimiento. </li><li> Etched andamios de PLGA se incubaron entonces en SBF a 37 ° C dentro de una incubadora con camisa de agua durante 12 horas, seguido de Mg 2 + y HCO 3 - SBF libre 2 por otro hr 12 a 37 ° C bajo agitación suave. </li><li> Andamios recubiertos de PLGA se enjuaga suavemente con estéril ddH 2 O para lavar el exceso de solución de cloruro de sodio, se secó en una campana de flujo laminar y desinfectado con 70% de etanol. </li><li> Integridad del recubrimiento de apatita se analizó con un microscopio electrónico de barrido (SEM). Recubiertos con apatita scaffolds fueron montados en los talones de SEM (Ted Pella) y recubiertos con carbono para mejorar la conductividad. El modo de electrones secundario se aplicó durante SEM (FEI / Phillips XL-30) observación. Dispersión de energía de rayos X del espectro se obtuvo a confirmar la composición elemental de las estructuras de apatito. </li></ol></li></ol> 3. Siembra Celular <ol><li> Al llegar a los niveles de sub-confluencia, se lavan las células con PBS (x2) y Tripsinizar. </li><li> Contar las células para cuantificar para la siembra </li><li> Semilla en precortadas andamios de diámetro 4,0 mm con 1,5 x 10 5 células. <ol class="ualpha"><li> Lugar individual andamio en un pocillo de una placa de 96 pocillos </li><li> Volver a suspender las células en medio de cultivo regulares con una concentración de aproximadamente 1,5 x 10 5 células por 20 l de medios de comunicación </li><li> Pipetear 20 l directamente sobre el andamio y el lugar en el cultivo celular incubadora durante 30 min. </li><li> Después de 30 min, añadir 200 l de medios de comunicación y permitir que las células se incubanen el andamio durante la noche antes de la cirugía </li><li> No es necesario para asegurar la adhesión de las células en el andamiaje como la siembra de un número suficiente de células se asegurará de que la mayoría se adhieren al andamio. Esto se ha validado en nuestro laboratorio con bioluminiscencia in vivo y análisis histológico. </li></ol></li></ol> 4. Creación de defectos de calota y la implantación in vivo <ol><li> Anesthetize adultos (60 días de edad) CD-1 de ratones desnudos con cóctel anestésico. (Ketamina / xilazina / acepromazina) a 50% de concentración (1:1 dilución con 0,9% de NaCl) o por régimen recomendado anestésico por protocolos de la institución. Estos ratones son atímicos para los hASCs no provocará una reacción inmune. <ol class="lalpha"><li> Dosificación: El clorhidrato de ketamina (80 mg / kg), xilazina (2,5 mg / kg) acepromazina (2,5 mg / kg) </li><li> Duración de acción: aproximadamente 30 min </li></ol></li><li> Quirúrgicamente cubrir el animal y esterilizar el quirúrgicositio con Betadine y alcohol (x3) y cubrir los ojos de ratón con pomada antes de hacer una incisión sagital en la línea media del cuero cabelludo del animal para exponer el hueso parietal derecho. Retire el pericráneo del hueso parietal derecho con raspado romo (fig. 1B). </li><li> Crear una unilateral de 4 mm de espesor completo defecto en el derecho no sutura del hueso parietal asociada mediante una estéril con recubrimiento de diamante broca trépano. Debe tener mucho cuidado de no alterar la duramadre subyacente (Figura 1B) como el espesor del ratón calota es &lt;0,3 mm. </li><li> Antes de la implantación, andamios enjuague con PBS estéril para evitar la transferencia de factores de crecimiento derivados de medio. </li><li> Coloque el andamio en el defecto. </li><li> Por último, suturar la piel se cerró y vigilar el animal por los protocolos establecidos postoperatorias. </li><li> Utilice analgesia establecido según las indicaciones de los protocolos de cuidado de los animales, según sea necesario después de la operación-por ejemplo. buprenorfina 0,1 mg / kg. </li></ol> 5. La cuantificación de la formación de osteoide <ol><li> Después de MicroCT se realizó en el ratón, la imagen tridimensional fue reconstruido utilizando software MicroView como se ha descrito anteriormente 1. </li><li> Uso de Adobe Photoshop, las imágenes eran de tamaño a una altura normal. </li><li> Con la función Magic Wand, los píxeles se mide el defecto craneal. </li><li> Porcentaje de curación del defecto se determinó mediante posteriores TC medir el área del defecto craneal y determinar el número de píxeles y dividiéndolo por el número de píxeles del defecto original </li><li> Por consiguiente, si el MicroCT no está disponible, una alternativa es usar Adobe Photoshop y cosecha de la bóveda craneal en puntos de tiempo determinados para histología. </li><li> Utilizando tinciones histológicas tales como azul de anilina y Pentacromo que denotan la formación de osteoide, múltiples secciones a lo largo de la duración del defecto debería ser manchado </li><li> Uso de Adobe Photoshop, afloran todas las áreas que no sonen el defecto craneal </li><li> Utilice la función varita mágica y determinar el número de píxeles de la formación de hueso de novo en la zona del defecto y comparar con los controles u otras variables. </li></ol> 6. Los resultados representativos El tejido adiposo tiene el potencial de servir un papel vital en la generación de células progenitoras para su aplicación clínica. El tejido adiposo tiene una ventaja única en que existe un suministro fácilmente disponible que puede ser recolectada en un procedimiento relativamente simple que implica un mínimo de morbilidad y mortalidad. Una vez que el tejido se recogieron y se recogió, nuestro protocolo se describe en la Figura 2. Adiposas derivadas de las células estromales se aíslan mediante una serie de pasos de lavado, digestión con colagenasa, y la centrifugación. Una vez que las células se sembraron en placas de cultivo, que se puede ampliar, se colocaron en diferentes protocolos de diferenciación in vitro, o se coloca directamente in vivo. El creación de la 4 mm de tamaño crítico defecto craneal proporciona un fácil acceso y reproducible en modelo in vivo para probar la capacidad de diferenciación osteogénica de nuestros derivadas de tejido adiposo células estromales. Mediante el uso de MicroCT, que son capaces de seguir la formación de tejido esquelético in vivo y el seguimiento del progreso de nuestras intervenciones (Figura 3). El defecto fundamental del calvario de tamaño no se cura dentro de la vida del animal y vemos aproximadamente el 90% de los defectos siguen siendo patente en torno a 8 semanas. El propio andamio (véase la discusión) tiene osteogénicas propiedades inductivas y ha demostrado la capacidad de inducir algunos de regeneración ósea. Los resultados típicos de colocación de andamio sin células muestra que alrededor de un tercio del defecto tendrá de novo la formación de hueso a las 8 semanas. Con el aumento de adiposas derivadas de las células del estroma, completamente dos tercios o más del defecto se muestran en la regeneración ósea alrededor de 8 semanas, aunque hay variability entre cada animal, y la cirugía. La formación de tejido esquelético se puede cuantificar mediante histología y los resultados típicos muestran una mayor formación de osteoide en las muestras con las ASC a través de la tinción de azul de anilina y Pentacromo (Figura 3C). Además, mostramos que las células implantadas humanos contribuyen a la formación de novo subyacente óseo a través del uso de hASCs GFP-etiquetados que muestran tinción in vivo a las 2 semanas cerca de la zona de formación de hueso de novo en el defecto craneal (Figura 3D) . Además, usamos inmunohistoquímica con un anticuerpo específico humano para mostrar la supervivencia de células humanas y contribución en la zona del defecto (Figura 3E). <img alt="Figura 1" src="/files/ftp_upload/4221/4221fig1.jpg" /> Figura 1 A -. El lipoaspirado tiene dos capas. La capa superior contiene los adipocitos y la mayoría de la processes material celular, mientras que la capa inferior contiene la solución salina utilizada durante el procedimiento lipoaspiración. B - la creación del defecto craneal a través de una incisión de línea media sobre el pericráneo para aislar el hueso parietal derecho, posterior creación de un 4 mm de tamaño crítico defecto craneal sin interrupción de la duramadre subyacente. <img alt="Figura 2" src="/files/ftp_upload/4221/4221fig2.jpg" /> Figura 2. Visión general de la cosecha y la aplicación de lipoaspirado del aislamiento de adiposas derivadas de las células del estroma a su expansión, diferenciación y uso in vitro e in vivo. <img alt="Figura 3" src="/files/ftp_upload/4221/4221fig3.jpg" /> Figura 3. Un MicroCT-muestra en la curación in vivo del defecto de tamaño crítico de calota con la aplicación de las ASC a través de un hydroxyapatitie andamio (fila inferior). Los controles incluyen ningún andamio y el defecto (fila superior) y el defecto con la colocación del andamio sin células (fila del medio) B - Cuantificación de la curación ósea a partir de la MicroCT mostrando curación significativamente mayor en el grupo de ASC. C - Histología mostrando una mayor formación de osteoide del grupo ASC (fila inferior) a través de la tinción de azul de anilina y Pentacromo. Para Anilina Azul, las manchas de color azul oscuro y osteoide por Pentacromo, las manchas osteoide amarillo. D - hASCS marcadas con GFP se sembraron en un andamio y se coloca en un defecto de la bóveda craneal y se sacrificaron a las 2 semanas. La tinción se realizó con una GFP anticuerpo marcado para mostrar las células humanas que contribuyen a la regeneración en la zona del defecto. E -. Inmunohistoquímica Humanos antígeno nuclear que muestra la prevalencia de las células humanas en el área del defecto a las 2 semanas <a href="http://www.jove.com/files/ftp_upload/4221/4221fig3large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .

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No hay conflictos de interés declarado.

Desde el aislamiento de tejido adiposo derivado de células estromales 2 de lipoaspirado, estas células se han diferenciado en una amplia variedad de linajes celulares. El tejido adiposo es de origen mesodérmico y por lo tanto, multipotentes derivadas de tejido adiposo células estromales será probablemente más eficaz con aplicación hacia un linaje mesodérmico. La capacidad de generar tejido óseo es especialmente importante dada la escasez de zonas donantes para un autoinjerto y las limitaciones inhere...

<table border="1"><tbody><tr><td> Nombre del reactivo: </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td></td><td></td><td></td><td> Lipoaspirado Harvest </td></tr><tr><td> PBS </td><td> Gibco </td><td> 10010-023 </td><td></td></tr><tr><td> Solución salina equilibrada de Hank </td><td> Cellgro </td><td> 21-023 CV- </td><td></td></tr><tr><td> La colagenasa </td><td> Sigma </td><td> C6885-500MG </td><td></td></tr><tr><td> Filtro de células 100 micras </td><td> BD Falcon </td><td> 352360 </td><td></td></tr><tr><td> Steri-top 500 ml 0,22 micras filtro </td><td> Millipore </td><td> SCGPT05RE </td><td></td></tr><tr><td></td><td></td><td></td><td> Defecto del calvario </td></tr><tr><td> Z500 Brushless MicromotorsUM50C </td><td> NSK </td><td> NSKZ500 </td><td></td></tr><tr><td> Circular Knife 4,0 mm </td><td> Xemax quirúrgica </td><td> CK40 </td><td></td></tr></tbody></table>

repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate

Reparación y regeneración del esqueleto craneofacial ofrece la promesa de la formación de tejido de novo a través de un enfoque basado en células utilizando células madre. Derivadas de tejido adiposo células del estroma (ASCs) han demostrado ser una fuente abundante de células madre multipotentes capaces de experimentar diferenciación osteogénica, condrogénicos, adipogénicos, y miogénica. Muchos estudios han explorado el potencial osteogénico de estas células in vivo con el uso de biomateriales andamios diferentes para la entrega celular. Se ha demostrado que mediante la utilización de un osteoconductivos, con recubrimiento de hidroxiapatita poli (láctico-co-glicólico) (PLGA-HA) andamio se sembró con ASC, un defecto de la bóveda craneal de tamaño crítico, un defecto que se define por su incapacidad de someterse espontánea cicatrización durante la vida del animal, se puede mostrar efectivamente la regeneración ósea robusta. Este modelo in vivo demuestra la base de enfoques translacional destinada a regenerar el tejido óseo - el celularcomponente y la matriz biológica. Este método sirve como un modelo para la aplicación clínica final de una célula progenitora hacia la reparación de un defecto específico de tejido.

Watch this Scientific Journal Video about Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate at JoVE.com

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Este protocolo describe el aislamiento de tejido adiposo derivado de células estromales de lipoaspirado y la creación de un 4 mm de tamaño crítico defecto craneal para evaluar la regeneración esquelética.

La reparación de un modelo de defecto crítico del tamaño de calota Uso adiposo derivados de las células del estroma cosechadas desde lipoaspirado

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

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15.8K vistas.  Stanford University. Este protocolo describe el aislamiento de tejido adiposo derivado de células estromales de lipoaspirado y la creación de un 4 mm de tamaño crítico defecto craneal para evaluar la regeneración esquelética.

Video: Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

Generación experimental de carcinoma asociada a los fibroblastos (CAF) a partir de fibroblastos humanos mamarios

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Investigación

Medicina

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Un modelo de la contundente unilateral Lesión Medular Cervical Uso del Impactador Infinite Horizon

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

Creación de fracturas rígidamente estabilizados para la evaluación de osificación intramembranosa, la distracción osteogénica, o la curación de los defectos críticos de tamaño

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Un modelo murino Nueva endovascular de aneurismas aórticos

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Un ratón fetal Modelo de la piel de la reparación de heridas sin cicatrices

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

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 ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Creación y el transplante de una hoja de células madre derivadas de adiposo (ASC) en un modelo de curación de herida diabética

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

Aislamiento de células regenerativas derivadas de tejido adiposo para el tratamiento de la disfunción eréctil después de la prostatectomía radical

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Un modelo de ratón BALB/c neonatal de enterocolitis necrosante

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...