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 hidrodinâmica de sRANKL DNA MC <ol><li> Entrega hidrodinâmica via mouse cauda Veia <ol><li> Pesar o mouse antes da injeção veia da cauda. Diluir sRANKL ou proteína fluorescente verde (GFP) de MC em solução de Ringer (pré-aquecer a 37 ° C) em um volume total de ~ 10% do peso do corpo do rato. </li><li> Aqueça o rato em uma gaiola por 10 min antes da injeção, a fim de dilatar os vasos sanguíneos e fazer nervuras laterais (VEs) visíveis. Monitorar o mouse com cuidado para evitar a desidratação e hipertermia. </li><li> Assim que os VEs estão dilatados e visíveis, transferir o mouse para o limitador e insira a ficha longe o suficiente para o barril para conter o movimento. Monitorar o mouse para a atividade normal, como respirar. Ajuste a ficha do limitador, se necessário. </li><li> Desinfectar a zona da injecção de 3/4 a partir da ponta da cauda com um algodão embebido em álcool. Use uma agulha G 27-30 para mouse injeção veia da cauda. Segure a cauda firmemente com uma mão e inserir a agulha emna veia da cauda. Aplique pressão na seringa e completar a injeção dentro de 5-7 seg. </li><li> Retire a agulha da veia da cauda e aplicar pressão com uma bola de algodão no local da injeção para parar o sangramento. Monitorar o mouse por 15-30 min, em seguida, transferir o mouse de volta ao viveiro uma vez que a taxa de respiração é reduzida para a taxa normal. </li></ol></li><li> A confirmação da sistêmica Rato sRANKL Expressão e Quantificação de Rato Serum TRACP5b Mensagem Gene Transferência <ol><li> Aqueça o rato em uma gaiola por 5-10 min, a fim de dilatar os vasos sanguíneos e fazer LVs visível. Monitorar o mouse com cuidado para evitar a desidratação e hipertermia. </li><li> Fazer uma pequena incisão com uma lâmina de cauda do rato num sítio uma frente a partir da ponta da cauda. </li><li> Colete aproximadamente 100 mL de sangue em tubos de separação de soro. </li><li> Incubar os tubos de separação de soro à temperatura ambiente durante 30 min. </li><li> Centrifugar as amostras a 10.000 xg durante 5 minutos e recolher soro. Store soro de -80 ° C para posterior análise. </li><li> Realizar sRANKL ELISA e ensaio do rato TRACP5b soro em amostras de soro usando um kit comercial disponível. </li></ol></li></ol> 2. Geração in vitro de osteoclastos de rato BMMs <ol><li> O isolamento e cultura de BMMs <ol><li> Isolamento da tíbia e os ossos do fêmur: Eutanásia o mouse doador com CO2, e desinfectar as pernas com etanol 70%. Dissecar a partir do osso púbico até o osso calcâneo, para remover o fêmur ea tíbia ossos intactos. </li><li> Retirar a pele e músculo cuidadosamente sem danificar o osso. Coloque fémur e tíbia processada em uma placa de Petri contendo tampão de fosfato salina (PBS). </li><li> Remoção de medula óssea: Corte a ponta de um lado do fêmur e da tíbia ossos e expulsar da medula óssea em um tubo de 50 ml utilizando uma seringa de 1 ml com uma agulha de 25 G, carregado com α-MEM contendo 1% de penicilina / estreptomicina (Osteoclasto Meio de Cultura / OCM). </li><li&gt; Processo de medula óssea: Misturar e transferir a suspensão de medula óssea para um novo tubo de 50 ml por passagem através de um filtro de células de 70 mM de nylon. Centrifugar as células a 300 x g e re-suspensão em α-MEM contendo 1% de penicilina / estreptomicina e 10% de SBF (meio de cultura de osteoclastos / OCM (+)). </li></ol></li><li> Culturas de macrófagos <ol><li> Contar as células usando um hemocitômetro ou contador automático de células. </li><li> Placa 1 x 10 6 culas por po para uma placa de 6 poços ou 3 x 10 5 células de lamelas de vidro (5 mm) ou fatias de dentina colocadas em placa de 96 poços e incuba-se a 37 ° C. </li><li> Aspirar todo o meio contendo células não aderentes e as células incubar a 37 ° C com nova OCM (+) contendo 25 ng / ml de rato M-CSF durante 48 horas. </li><li> Aspirar todos os meios e incubar as células a 37 ° C, com fresco OCM (+) mídia contendo 25 ng / ml rato M-CSF, e 30 ng / ml sRANKL mouse para iniciar a osteoclastogênese. Reabastecer media a cada 3 dias, conforme necessário. </li><li> Cultivar célulasdurante aproximadamente 6-8 dias, para formar células gigantes multi-nucleares capazes de reabsorção óssea. Assim que as células multinucleadas aparecer, execute TRAP (fosfatase ácida tartarato resistente) coloração utilizando um kit comercial disponível, e quando totalmente amadurecido, realizar ensaios funcionais. Visualize anéis F-actina em lamelas usando mancha phalloidin e imagem das células aderidas à dentina fatias por microscopia eletrônica de varredura (SEM), como descrito anteriormente 19. </li></ol></li></ol> 3. Geração in vitro de osteoclastos a partir de PBMC humanas <ol><li> Isolamento de PBMC Humano <ol><li> Adicionar 10 ml de pré-aquecido de Histopaque-1077 para um tubo de 50 ml. </li><li> Filtro de leucócitos vazio obtido a partir do banco de sangue com PBS estéril para um tubo de 50 mL (proporção de 1:1 de sangue com PBS). Camada do sangue diluído sobre a solução histopaque muito lentamente, certificando-se de que o sangue não se mistura com o histopaque. </li><li> Centrifugar as amostras a 1000 xg durante 20 min a 18 ° C. Seguiring centrifugação, cuidadosamente isolar a camada buffy coat branco que contém as células brancas do sangue e transferir para um novo tubo de 50 ml. </li><li> Dilui-se as células com PBS e centrifugar novamente a 650 xg durante 5 min para recolher as células. </li><li> Remover o sobrenadante e ressuspender as células em OCM (-) media e contagem de células utilizando contador de células. </li></ol></li><li> Chapeamento e cultivando PBMCs <ol><li> Ressuspender as células em OCM (+) e meios de placa de 8 x 10 6 células / ml por poço numa placa de 6 poços ou de 1 x 10 6 células / ml por poço em placas de 96 poços. Células da placa em lamelas de vidro (5 mm), tal como exigido para imagiologia por imunofluorescência, bem como em fatias de osso adequadas para ensaios de reabsorção óssea, como previamente descrito 20. </li><li> Alterar meios de cultura, repondo as células com M-CSF humano a cada 2-3 dias ou quando necessário. Em seguida, avance para o passo de diferenciação no dia 5 pela adição de 30 ng / ml de sRANKL humano junto com 25 ng ml M-CSF humano / no OCM (+) de mídia para iniciar osteoclastogenesé. </li><li> Cultivar células em, aproximadamente, 14 a 21 dias para formar células gigantes multi-nucleares. Assim que as células multinucleadas aparecer, estas células podem então ser marcados de TRAP, e quando totalmente amadurecido, ensaios funcionais podem ser realizadas. Anéis de F-actina pode ser visualizado em lamelas utilizando faloidina mancha. A morfologia das células pode ser examinado em células ligadas à dentina fatias por SEM. </li></ol></li></ol>

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The authors have nothing to disclose.

Condições músculo-esqueléticas são as principais causas de morbidade e incapacidade e são compostas de mais de 150 doenças e síndromes; afetando cerca de 90 milhões de americanos hoje. Inflamação das articulações e destruição óssea são características predominantes de doenças músculo-esqueléticas, incluindo artrite e osteoporose. A osteoporose é uma condição que enfraquece a integridade óssea, muitas vezes levando a fraturas do osso. A artrite é uma doença crônica, debilitante doença caracte...

Doenças músculo-esqueléticas afectam milhões de pessoas nos Estados Unidos e apresentar conseqüências graves para os sistemas nacionais e locais de saúde 1. Estes distúrbios são caracterizados por perda de massa óssea e da função articular que requerem tratamento extensivo e longos períodos de recuperação. Vulgarmente, um aumento relativo no número e / ou actividade dos osteoclastos, as células especializadas para reabsorver osso, na osteoporose e artrite observadas é 2. Sob condições fisiológicas, o número e a actividade dos osteoclastos é regulada pelo activador do receptor do factor nuclear κ-B ligando (RANKL), o qual é produzido por osteoblastos. A osteoprotegerina (OPG), um receptor de engodo para RANKL também é produzido por osteoblastos três modelos animais in vivo que envolvem a sobre-expressão sistémica de sRANKL, ou deleção de OPG são muito valiosas na pesquisa de osteoporose.; No entanto, estes métodos requerem a geração de ratinhos transgénicos 4,5. Aqui, uma nova alternativaMétodo de overexpressing sRANKL para o estudo de desordens relacionadas com musculoesqueléticas é descrito. Especificamente, minicírculo (MC) a tecnologia de DNA e métodos de entrega hidrodinâmicas foram utilizadas para conseguir a transferência do gene de sRANKL in vivo e sobre-expressar rato sRANKL sistemicamente 6. Este método também é complementar a outros modelos in vivo de osteoporose, tais como a modulação hormonal de osteoclastos seguintes ovariectomia 7 e a intervenção dietética com uma dieta pobre em cálcio 8. Estes modelos são muito úteis para estudar os diferentes aspectos de distúrbios osteomusculares relacionados ao porém eles necessitam de procedimentos cirúrgicos e pode demorar até vários meses, a um custo significativo 9. Ovariectomizadas (OVX) modelo de roedor é um modelo animal experimental em que a remoção dos ovários leva à deficiência de estrogênio imitando assim osteoporose pós-menopausa humana 10. Human osteoporose pós-menopausa, uma condição em que o estrogênio Deficiparência leva ao aumento do risco de fraturas ósseas e osteoporose afeta cerca de oito milhões de mulheres nos Estados Unidos. Embora o modelo de OVX é útil para a osteoporose pós-menopausa que oferece vantagens limitadas em estudar a osteoporose em geral. Estrogen suprime a perda óssea, induzindo a inibição de osteoclastos e osteoblastos apoptose, portanto, na sua ausência de um aumento da actividade dos osteoclastos é observada 10-12. A relação de desequilíbrio RANKL-OPG que favorece a reabsorção óssea também é observada 13. No entanto, a deficiência de estrogénio, in vivo, também é acompanhada por uma diminuição dos níveis de factor de crescimento transformante β (TGF β), o aumento da interleucina-7 (IL-7) e de TNF, IL-1 e IL-6, 14,15. Como estas citocinas conhecidas funções moduladores de remodelação óssea independente da via de RANKL, é impossível atribuir qualquer activação dos osteoclastos unicamente ao eixo RANKL-RANK. O modelo descrito neste trabalho permite aos pesquisadores estudar em vivo eixo RANKL-RANK na osteoclastogênese e perda óssea sem citocinas pró-inflamatórias em comparação com modelos de roedores OVX. Além disso, técnicas in vitro osteoclastogênese são ferramentas essenciais para estudar a ativação dos osteoclastos para potenciais tratamentos terapêuticos de doenças músculo-esqueléticas. Estudos anteriores também mostraram que a cultura de medula óssea do rato macrófagos derivados (BMMs) com o mouse macrófagos fator estimulador de colônias (M-CSF) e mouse sRANKL pode levar a diferenciação de osteoclastos 3,16,17. Aqui, os protocolos de produção de células semelhantes a osteoclastos multinucleadas da medula óssea de rato, bem como a partir de células mononucleares do sangue periférico humano (PBMCs) in vitro, 18 encontram-se descritos. Os ensaios baseados em células necessárias para definir um osteoclasto terminalmente diferenciadas e totalmente funcional madura também são descritas resumidamente. Estas técnicas in vitro complementar o romance em abordagem vivo e servem como pinstrumentos de investigação owerful para estudar a diferenciação dos osteoclastos e ativação. Utilizando estes sistemas, os cientistas são capazes de gerar os osteoclastos in vivo e in vitro e definir os estímulos e sinais necessários para a sua proliferação e activação, assim como testar a eficácia dos inibidores farmacológicos e 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.

Aqui, uma técnica de transferência de gene novo para a diferenciação de osteoclastos in vivo e os protocolos de cultura de células para a diferenciação de células precursoras em osteoclastos in vitro, como um método para estudar os efeitos das citocinas em osteoclastogénese são descritos. Na Figura 1, os resultados representativos de transferência de gene bem sucedida da GFP e rato sRANKL MC em ratinhos são mostrados. Na Figura 2, as imagens representativa...

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 Gene Transferência e<em&gt; In vitro</em&gt; Ensaios baseados em células para o Estudo da perda óssea em Distúrbios Osteomusculares

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

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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 visualizações.  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. 

Vídeo: 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.

Tratamento de drogas e<em&gt; In Vivo</em&gt; Imagem de Osteoblast-osteoclastos Interações em um modelo de Medaka Peixe Osteoporose

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

Biologia do Desenvolvimento

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.

Modelos de metástase óssea

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.

Seqüente<em&gt; In vivo</em&gt; Imagem de osteogênicas células-tronco / progenitoras durante o reparo de fratura

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; In vivo</em&gt; Optical e μCT imagem para monitorar infecções, inflamações, and Bone Anatomy em uma infecção Orthopaedic Implant em Ratos

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.

aplicações de<em&gt; In Vivo</em&gt; Teste Funcional do Rato tibial anterior para a Avaliação da engenharia de tecidos Repair Músculo Esquelético

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

Bioengenharia

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.

Modelo Câmara fêmur Janela para<em&gt; In Vivo</em&gt; Acompanhamento de celular na Murino de Medula Óssea

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

Imunologia e Infecção

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.

Semiautomated Longitudinal baseados em Microcomputed tomografia computadorizada quantitativa análise estrutural de um modelo de fratura Vertebral relacionadas com osteoporose rato Nude

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>Análise multimodal da formação óssea usando modelos de explante

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 em destaque: Técnicas avançadas para caracterizar a mineralização de tecidos na pesquisa de regeneração óssea

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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Caracterização abrangente da mineralização tecidual em um modelo ex vivo

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