Name: a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders
Uploaded: 2014-06-08T23:30:00.000+00:00
Duration: 11 min 47 s
Description: Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

Research was partly supported by NIH research grants R01 AR062173 and SHC 250862 to IEA. ES is the recipient of NIH T32 CTSC predoctoral fellowship.

1. Entrega hidrodinámico de ADN MC sRANKL <ol><li> Entrega hidrodinámica a través del ratón de la cola de la vena <ol><li> Pesar el ratón antes de la inyección en la vena de la cola. Diluir sRANKL o proteína fluorescente verde (GFP) MC en solución de Ringer (pre-calentamiento a 37 ° C) en un volumen total de ~ 10% del peso corporal del ratón. </li><li> Caliente el ratón en una jaula durante 10 min antes de la inyección con el fin de dilatar los vasos sanguíneos y hacer que las venas laterales (LVs) visibles. Monitorear el ratón con cuidado para evitar la deshidratación y la hipertermia. </li><li> Tan pronto como los volúmenes lógicos están dilatados y visibles, transferir el ratón a la inmovilización e inserte el conector lo suficiente en el barril para restringir el movimiento. Monitorear el ratón para la actividad normal, como la respiración. Ajuste el tapón de inmovilización, si es necesario. </li><li> Desinfectar el área de la inyección de 3/4 de la punta de la cola con un algodón con alcohol. Utilice una aguja de 27 a 30 para el ratón inyección en la vena de la cola. Sostenga la cola firmemente con una mano e inserte la aguja ena la vena de la cola. Aplique presión sobre la jeringa y completar la inyección dentro de 5-7 seg. </li><li> Retire la aguja de la vena de la cola y aplique presión con un algodón sobre el lugar de la inyección para detener el sangrado. Monitorear el ratón durante 15-30 minutos, a continuación, transferir el ratón de nuevo a vivarium una vez que el ritmo de la respiración se reduce a su ritmo normal. </li></ol></li><li> Confirmación de la expresión y cuantificación de Ratón Serum TRACP5b Mensaje Gene Transfer sistémica Ratón sRANKL <ol><li> Caliente el ratón en una jaula durante 5-10 minutos con el fin de dilatar los vasos sanguíneos y hacer LV visible. Monitorear el ratón con cuidado para evitar la deshidratación y la hipertermia. </li><li> Hacer una pequeña incisión con una cuchilla a la cola del ratón en un sitio de una cuarta parte de la punta de la cola. </li><li> Recoge alrededor de 100 l de sangre en tubos de separación de suero. </li><li> Incubar los tubos de separación de suero a temperatura ambiente durante 30 min. </li><li> Centrifugar las muestras a 10.000 xg durante 5 min y recoger el suero. Store suero en -80 ° C para su posterior análisis. </li><li> Realizar sRANKL ELISA y ensayo de TRACP5b suero de ratón en muestras de suero utilizando un kit comercial disponible. </li></ol></li></ol> 2. In vitro generación de osteoclastos de Ratón BMMs <ol><li> Aislamiento y cultivo de BMMs <ol><li> El aislamiento de la tibia y los huesos de fémur: La eutanasia el ratón donante con CO 2, y desinfectar las piernas con etanol al 70%. Diseccionar desde el pubis hasta el hueso calcáneo, para quitar los huesos fémur y la tibia intacta. </li><li> Quite la piel y el músculo cuidadosamente sin dañar el hueso. Coloque fémur procesado y hueso de la tibia en una placa de Petri que contiene tampón fosfato salino (PBS). </li><li> La eliminación de la médula ósea: Corte la punta en un lado de los huesos del fémur y de la tibia y enjuagar la médula ósea en un tubo de 50 ml utilizando una jeringa de 1 ml con una aguja de calibre 25, cargado con α-MEM que contiene 1% de penicilina / estreptomicina (osteoclastos Cultura Medio / OCM). </li><li&gt; Proceso de médula ósea: Mezclar y transferir la suspensión de la médula ósea a un nuevo tubo de 50 ml pasando a través de un filtro de células de 70 m de nylon. Centrifugar las células a 300 xg y volver a suspender en α-MEM que contiene 1% de penicilina / estreptomicina y 10% de FBS (medio de osteoclastos Cultura / OCM (+)). </li></ol></li><li> Cultura de los macrófagos <ol><li> Contar las células utilizando un hemocitómetro o un contador automático de células. </li><li> Plate 1 x 10 6 células por pocillo en una placa de 6 pocillos o 3 x 10 5 células a cubreobjetos de vidrio (5 mm) o rodajas de dentina colocados en placas de 96 pocillos y se incuba a 37 ° C. </li><li> Aspirar todos los medios que contienen las células no adherentes y se incuban las células a 37 ° C con OCM fresca (+) que contiene 25 ng / ml ratón M-CSF durante 48 horas. </li><li> Aspirar todos los medios de comunicación y se incuban las células a 37 ° C con frescos OCM (+) un medio que contenía 25 ng / ml ratón M-CSF, y 30 ng / ml sRANKL ratón para iniciar la osteoclastogénesis. Reponer los medios de comunicación cada 3 días según sea necesario. </li><li> Se cultivan las célulasdurante aproximadamente 6-8 días para formar células multinucleares gigantes capaces de resorción ósea. En cuanto aparezcan células multinucleadas, realice TRAP (fosfatasa ácida resistente al tartrato) tinción utilizando un kit comercial disponible, y cuando está completamente madura, realizar ensayos funcionales. Visualizar anillos de actina F en cubreobjetos utilizando mancha faloidina y la imagen de las células unidas a la dentina rebanadas por microscopía electrónica de barrido (SEM) como se ha descrito previamente 19. </li></ol></li></ol> 3. Generación de osteoclastos in vitro de las PBMC humanas <ol><li> Aislamiento de PBMC humanas <ol><li> Añadir 10 ml de pre-calentado Histopaque-1077 en un tubo de 50 ml. </li><li> Filtro de leucocitos vacío obtiene a partir del banco de sangre con PBS estéril en un tubo de 50 ml (proporción de 1:1 de sangre con PBS). Capa de la sangre diluida sobre la solución Histopaque muy lentamente, asegurándose de que la sangre no se mezcla con el histopaque. </li><li> Centrifugar las muestras a 1000 xg durante 20 min a 18 ° C. Seguiring centrifugación, aislar cuidadosamente la capa de la capa leucocitaria blanca que contiene las células blancas de la sangre y transferir a un nuevo tubo de 50 ml. </li><li> Diluir las células con PBS y se centrifuga de nuevo a 650 xg durante 5 minutos para recoger las células. </li><li> Aspirar el sobrenadante y volver a suspender las células en OCM (-) de los medios y contar las células utilizando un contador de células. </li></ol></li><li> Plating y cultivo de CMSP <ol><li> Resuspender las células en las OCM (+) los medios de comunicación y la placa de 8 x 10 6 células / ml por pocillo en una placa de 6 pocillos o 1 x 10 6 células / ml por pocillo en placas de 96 pocillos. Las células de la placa sobre cubreobjetos de vidrio (5 mm) según se requiera para obtener imágenes de inmunofluorescencia, así como en secciones de hueso adecuados para ensayos de resorción ósea como anteriormente descritos 20. </li><li> Cambio de los medios de cultivo, la reposición de las células con M-CSF humano cada 2-3 días o según sea necesario. A continuación, proceder a la etapa de diferenciación en el día 5 por la adición de 30 ng / ml de sRANKL humana junto con 25 ml de M-CSF ng / humano en OCM (+) medios para iniciar osteoclastogeneses. </li><li> Se cultivan las células durante aproximadamente 14 a 21 días para formar células multinucleares gigantes. Tan pronto como aparecen células multinucleadas, a continuación, estas células se pueden etiquetar para TRAP, y cuando está completamente madurado, ensayos funcionales pueden llevarse a cabo. Anillos de actina F se pueden visualizar en cubreobjetos utilizando tinción phalloidin. La morfología de las células puede examinarse en células unidas a la dentina rebanadas por SEM. </li></ol></li></ol>

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The authors have nothing to disclose.

Las afecciones musculoesqueléticas son las principales causas de morbilidad y discapacidad, y se componen de más de 150 enfermedades y síndromes; que afecta hoy a unos 90 millones de estadounidenses. Inflamación de las articulaciones y la destrucción ósea son las características predominantes de los trastornos musculoesqueléticos, como la artritis y la osteoporosis. La osteoporosis es una enfermedad que debilita la integridad ósea, a menudo conduce a las fracturas de los huesos. La artritis es una enfermedad cr...

Enfermedades musculoesqueléticas afectan a millones de personas en los Estados Unidos y del presente graves consecuencias para los sistemas de salud nacionales y locales 1. Estos trastornos se caracterizan por la pérdida de hueso y función de las articulaciones que requieren tratamiento extenso y largos períodos de recuperación. Comúnmente, un aumento relativo en el número y / o actividad de los osteoclastos, células especializadas para reabsorber el hueso, en la osteoporosis y la artritis se observa 2. En condiciones fisiológicas el número y la actividad de los osteoclastos está regulada por el activador del receptor del factor nuclear κ-B ligando (RANKL), que es producida por los osteoblastos. La osteoprotegerina (OPG), un receptor señuelo para RANKL también es producida por los osteoblastos 3 modelos animales in vivo que implican la sobreexpresión sistémica de sRANKL, o supresión de OPG son muy valiosa en la investigación de la osteoporosis.; Sin embargo, estos métodos requieren la generación de ratones transgénicos 4,5. Aquí, una nueva alternativamétodo de sobreexpresar sRANKL para el estudio de los trastornos musculoesqueléticos relacionados-se describe. Específicamente, minicircle (MC) la tecnología del ADN y los métodos de entrega hidrodinámicas se utilizaron para lograr la transferencia de genes de sRANKL in vivo e sobreexpresan ratón sRANKL sistémicamente 6. Este método también es complementaria a otros modelos in vivo de la osteoporosis, tales como la modulación hormonal de los osteoclastos siguientes ovariectomía 7 y la intervención dietética con dieta baja en calcio 8. Estos modelos son muy útiles para estudiar diferentes aspectos de los trastornos musculoesqueléticos relacionados-sin embargo, requieren de procedimientos quirúrgicos y pueden durar hasta varios meses, a un costo significativo 9. Ovariectomizadas (OVX) modelo de roedores es un modelo animal experimental en que la extirpación de los ovarios conduce a la deficiencia de estrógenos imitando así la osteoporosis posmenopáusica humana 10. La osteoporosis post-menopáusica humana, una condición donde Defici estrógenoparencia conduce a un mayor riesgo de fracturas óseas y la osteoporosis afecta a aproximadamente ocho millones de mujeres en los Estados Unidos solamente. Aunque el modelo OVX es útil para la osteoporosis post-menopáusica que ofrece ventajas limitadas en el estudio de la osteoporosis en general. El estrógeno suprime la pérdida de hueso, mediante la inducción de osteoclastos y la inhibición de la apoptosis de los osteoblastos, por lo tanto, en su ausencia se observa un aumento de la actividad de los osteoclastos 10-12. Un desequilibrio en la relación RANKL-OPG que favorece la resorción ósea también se observa 13. Sin embargo, la deficiencia de estrógenos en vivo también se acompaña de disminución de los niveles de transformación del factor de crecimiento β (TGF β), aumento de la interleucina-7 (IL-7) y de TNF, IL-1 e IL-6 14,15. Como estas citoquinas han conocido funciones moduladoras de remodelación ósea independiente de la vía de RANKL, es imposible atribuir cualquier activación de los osteoclastos exclusivamente al eje RANKL-RANK. El modelo que se describe en este documento permite a los investigadores estudiar en vivo eje RANKL a RANK en la osteoclastogénesis y la pérdida ósea sin las citoquinas pro-inflamatorias en comparación con modelos de roedores OVX. Además, las técnicas de la osteoclastogénesis in vitro son herramientas esenciales para el estudio de la activación de los osteoclastos para posibles tratamientos terapéuticos de las enfermedades musculoesqueléticas. Estudios anteriores también han demostrado que el cultivo de macrófagos derivados de médula ósea de ratón (BMMs) con macrófagos de ratón factor estimulante de colonias (M-CSF) y el ratón sRANKL puede conducir a la diferenciación de los osteoclastos 3,16,17. Aquí se describen los protocolos para generar células similares a osteoclastos multinucleadas de médula ósea de ratón, así como de células de sangre periférica humana (PBMC) in vitro 18. Los ensayos basados ​​en células necesarios para definir un osteoclastos terminales diferenciadas y completamente funcional madura también se describen brevemente. Estas técnicas in vitro complementan la novela en el enfoque vivo y juntos sirven como pherramientas de investigación para estudiar owerful diferenciación de los osteoclastos y la activación. El uso de estos sistemas, los científicos son capaces de generar los osteoclastos in vivo e in vitro y definir los estímulos y señales requeridas para su proliferación y activación, así como probar la eficacia de los inhibidores farmacológicos y biológicos.

<table><tbody><tr><td>alpha-MEM</td><td>Life Technologies&amp;nbsp;</td><td>12561-056</td><td></td></tr><tr><td>Human M-CSF</td><td>Miltenyi Biotec</td><td>130-096-492</td><td></td></tr><tr><td>Mouse M-CSF</td><td>Miltenyi Biotec</td><td>130-094-643</td><td></td></tr><tr><td>Human RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-631</td><td></td></tr><tr><td>Mouse RANK-Ligand - soluble</td><td>Miltenyi Biotec</td><td>130-094-076</td><td></td></tr><tr><td>Tailveiner Restrainer for mice</td><td>Braintree</td><td>TV-150 STD</td><td></td></tr><tr><td>Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>MTR00</td><td></td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma</td><td>387A</td><td></td></tr><tr><td>MouseTRAP assay&amp;nbsp;</td><td>immunodiagnostic systems</td><td>SB-TR103</td><td></td></tr></tbody></table>

a novel in vivo gene transfer technique and in vitro cell based assays for the study of bone loss in musculoskeletal disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

Aquí, una técnica de transferencia de gen nuevo para la diferenciación de los osteoclastos in vivo y protocolos de cultivo de células para la diferenciación de células precursoras en los osteoclastos in vitro como un método para estudiar los efectos de las citoquinas en la osteoclastogénesis se describen. En la Figura 1, se muestran los resultados representativos de la transferencia de genes exitosa de GFP y ratón sRANKL MC en ratones. En la Figura 2, se muest...

Watch this Scientific Journal Video about A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders at JoVE.com

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Técnica de transferencia de genes y<em&gt; In vitro</em&gt; ensayos basados ​​en células para el estudio de la pérdida ósea en los trastornos musculoesqueléticos

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

a-novel-vivo-gene-transfer-technique-vitro-cell-based-assays-for

Research

JoVE Journal

Medicine

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

11.6K vistas.  University of California, Davis. Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described. 

Video: A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

El tratamiento de drogas y<em&gt; En Vivo</em&gt; Imagen de osteoblasto-osteoclastos Interacciones en un modelo Medaka pescado osteoporosis

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Investigación

Biología del Desarrollo

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Los modelos de metástasis en los huesos

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Medicina

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Secuencial<em&gt; En vivo</em&gt; Imagen de células madre / progenitoras osteogénicas durante la reparación de la fractura

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinado<em&gt; En vivo</em&gt; Óptica y μCT imágenes para vigilar la infección, inflamación y Hueso anatomía en una infección de implantes ortopédicos en ratones

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Aplicaciones de la<em&gt; En Vivo</em&gt; Pruebas funcionales de la rata tibial anterior para la evaluación de la ingeniería tisular de reparación del músculo esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Bioingeniería

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Fémur Window Cámara Modelo de<em&gt; En Vivo</em&gt; Seguimiento de la célula de la médula ósea murina

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Inmunología e Infección

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

Semiautomatizado Longitudinal basado en la tomografía Microcomputed cuantitativo análisis estructural de un modelo de rata desnuda fractura Vertebral relacionada con la Osteoporosis

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

<meta charset="utf8"></head><body>Análisis multimodal de la formación ósea mediante modelos de explantes

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

<meta charset="utf8"></head><body>Autor destacado: Técnicas avanzadas para caracterizar la mineralización de tejidos en la investigación de la regeneración ósea

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

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Aplicaciones de la

Fémur Window Cámara Modelo de

Semiautomatizado Longitudinal basado en la tomografía Microcomputed cuantitativo análisis estructural de un modelo de rata desnuda fractura Vertebral relacionada con la Osteoporosis

Caracterización integral de la mineralización tisular en un modelo ex vivo

Caracterización integral de la mineralización tisular en un modelo ex vivo

Caracterización integral de la mineralización tisular en un modelo ex vivo