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. Livraison hydrodynamique de sRANKL MC ADN <ol><li> Livraison hydrodynamique via souris veine de la queue <ol><li> Peser la souris avant l&#39;injection dans la veine caudale. Diluer sRANKL ou la protéine fluorescente verte (GFP) dans MC solution de Ringer (pré-chauffer à 37 ° C) dans un volume total d&#39;environ 10% du poids corporel de la souris. </li><li> Réchauffez la souris dans une cage pendant 10 minutes avant l&#39;injection afin de dilater les vaisseaux sanguins et faire nervures latérales (EFV) visibles. Surveillez attentivement la souris pour éviter la déshydratation et d&#39;hyperthermie. </li><li> Dès que les VL sont dilatés et visibles, transférer la souris pour le dispositif de retenue et insérer la fiche assez loin dans le canon pour restreindre le mouvement. Surveillance de la souris pour les activités normales comme la respiration. Réglez la prise de contention si nécessaire. </li><li> Désinfecter la zone d&#39;injection 3/4 de la pointe de la queue avec un tampon imbibé d&#39;alcool. Utiliser une aiguille 27-30 G pour injection veine de la queue de la souris. Tenez la queue fermement d&#39;une main et insérer l&#39;aiguille dansà la veine de la queue. Appliquer une pression sur la seringue et compléter l&#39;injection dans 5-7 sec. </li><li> Retirez l&#39;aiguille de la veine de la queue et appliquer une pression avec une boule de coton sur le site d&#39;injection pour arrêter le saignement. Surveillance de la souris pendant 15-30 minutes, puis transférer la souris vers vivarium fois le taux de respiration est réduite à son taux normal. </li></ol></li><li> Confirmation de l&#39;expression et la quantification de souris systémique de transfert souris Sérum TRACP5b Poster Gene <ol><li> Réchauffez la souris dans une cage pendant 5-10 min afin de dilater les vaisseaux sanguins et faire VL visible. Surveillez attentivement la souris pour éviter la déshydratation et d&#39;hyperthermie. </li><li> Faire une petite incision avec une lame à la queue de la souris à un site d&#39;une suite de la pointe de la queue. </li><li> Recueillir environ 100 pi de sang dans des tubes de séparation de sérum. </li><li> Incuber les tubes de séparation de sérum à température ambiante pendant 30 min. </li><li> Centrifuger les échantillons à 10 000 g pendant 5 min et recueillir le sérum. Store sérum à -80 ° C pour analyse ultérieure. </li><li> Effectuer sRANKL ELISA et test de la souris TRACP5b de sérum sur des échantillons de sérum en utilisant un kit disponible dans le commerce. </li></ol></li></ol> 2. Ostéoclastes in vitro génération de BMMS souris <ol><li> Isolement et culture des BMMS <ol><li> L&#39;isolement du tibia et du fémur os: Euthanasier la souris donneuse avec du CO 2, et à désinfecter les jambes avec 70% d&#39;éthanol. Disséquer de l&#39;os pubien à l&#39;os du calcanéum, de supprimer fémur et du tibia os intact. </li><li> Retirez soigneusement la peau et les muscles sans endommager l&#39;os. Placez fémur et du tibia traitées os dans une boîte de Petri contenant un tampon phosphate salin (PBS). </li><li> Prélèvement de moelle osseuse: Couper le bout sur un côté du fémur et du tibia os et débusquer la moelle osseuse dans un tube de 50 ml à l&#39;aide d&#39;une seringue de 1 ml avec une aiguille 25 G, chargé de α-MEM contenant 1% de pénicilline / streptomycine (ostéoclastes milieu de culture / OCM). </li><li&gt; Procédé de moelle osseuse: mélanger et transférer la suspension de moelle osseuse pour un nouveau tube de 50 ml en la faisant passer à travers un tamis cellulaire en nylon de 70 pm. Centrifuger les cellules à 300 xg et remettre en suspension dans α-MEM contenant 1% de pénicilline / streptomycine et 10% de FBS (milieu ostéoclastes Culture / OCM (+)). </li></ol></li><li> Culture de macrophages <ol><li> Compter les cellules en utilisant un hématimètre ou compteur de cellules automatique. </li><li> Plate 1 x 10 6 cellules par puits pour une plaque à 6 puits ou 3 x 10 5 cellules de lamelles de verre (5 mm) ou des tranches de dentine placés en plaque de 96 puits et incuber à 37 ° C. </li><li> Aspirer tous les médias contenant des cellules non-adhérentes et incuber les cellules à 37 ° C avec frais OCM (+) contenant 25 ng / ml souris M-CSF pendant 48 heures. </li><li> Aspirer tous les médias et incuber les cellules à 37 ° C avec frais OCM (+) des milieux contenant 25 ng / ml souris M-CSF, et 30 ng / ml sRANKL de la souris pour lancer ostéoclastogénèse. Reconstituer des médias tous les 3 jours selon les besoins. </li><li> Cultiver des cellulespour environ 6-8 jours pour former des cellules multi-nucléaires géants capables de résorption osseuse. Dès que les cellules multinucléées apparaissent, effectuer TRAP (tartrate de la phosphatase acide résistant) coloration en utilisant un kit disponible dans le commerce, et à pleine maturité, effectuer des tests fonctionnels. Visualiser les anneaux F-actine sur des lamelles à l&#39;aide de la phalloïdine tache et de l&#39;image des cellules attachées à la dentine tranches par microscopie électronique à balayage (MEB), comme décrit précédemment 19. </li></ol></li></ol> 3. Ostéoclastes in vitro génération à partir de PBMC humaines <ol><li> Isolement de cellules PBMC humaines <ol><li> Ajouter 10 ml d&#39;Histopaque-1077 pré-chauffé dans un tube de 50 ml. </li><li> Filtre de leucocytes vide obtenu à partir de la banque de sang avec du PBS stérile dans un tube de 50 ml (rapport 1:1 de sang avec de la PBS). Couche du sang dilué sur la solution de histopaque très lentement, faire en sorte que le sang ne se mélange pas avec le histopaque. </li><li> Centrifuger les échantillons à 1000 g pendant 20 min à 18 ° C. Suivrement centrifugation, isoler soigneusement la couche leuco-plaquettaire blanc contenant les globules blancs et transférer dans un nouveau tube de 50 ml. </li><li> Diluer les cellules avec du PBS et on centrifuge à nouveau à 650 x g pendant 5 minutes pour recueillir les cellules. </li><li> Retirer le surnageant et remettre les cellules en OCM (-) les médias et compter les cellules en utilisant un compteur de cellules. </li></ol></li><li> Placage et la culture des PBMC <ol><li> Remettre en suspension les cellules dans l&#39;OCM (+) des médias et plateau de 8 x 10 6 cellules / ml par puits dans une plaque à 6 puits ou 1 x 10 6 cellules / ml par puits dans une plaque de 96 puits. Cellules de la plaque sur des lamelles de verre (5 mm) tel que requis pour l&#39;imagerie par immunofluorescence, ainsi que sur des tranches osseuses appropriées pour des dosages de la résorption osseuse tels que décrits précédemment 20. </li><li> Changer de milieux de culture, la reconstitution des cellules avec M-CSF humain tous les 2-3 jours ou au besoin. Ensuite, passez à l&#39;étape de différenciation au jour 5 par addition de 30 ng / ml de sRANKL humain avec 25 ml de M-CSF humain / ng dans OCM (+) médias pour lancer osteoclastogenesest. </li><li> Cultiver des cellules d&#39;environ 14 à 21 jours pour former des cellules multi-nucléaires géants. Dès que les cellules multinucléées apparaissent, ces cellules peuvent ensuite être étiquetés pour TRAP, et à pleine maturité, les essais fonctionnels peuvent être effectuées. Anneaux F-actine peuvent être visualisées sur des lamelles à l&#39;aide phalloidin tache. La morphologie des cellules peut être examinée dans des cellules attachées à la dentine tranches par SEM. </li></ol></li></ol>

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The authors have nothing to disclose.

Troubles musculo-squelettiques sont les principales causes de morbidité et d&#39;invalidité et sont constitués de plus de 150 maladies et syndromes; affectant environ 90 millions d&#39;Américains aujourd&#39;hui. L&#39;inflammation des articulations et la destruction osseuse sont des caractéristiques prédominantes de troubles musculo-squelettiques, dont l&#39;arthrite et l&#39;ostéoporose. L&#39;ostéoporose est une maladie qui fragilise l&#39;intégrité de l&#39;os, ce qui conduit souvent à des fractures de l&...

Maladies musculo-squelettiques affectent des millions de personnes aux Etats-Unis et actuelles de graves conséquences pour les systèmes nationaux et locaux de la santé 1. Ces troubles sont caractérisés par la perte de l&#39;os et la fonction articulaire qui nécessitent un traitement lourd et de longues périodes de récupération. Généralement, une augmentation relative du nombre et / ou l&#39;activité des ostéoclastes, cellules spécialisées à résorber l&#39;os, l&#39;ostéoporose et l&#39;arthrite est observée 2. Dans des conditions physiologiques, le nombre et l&#39;activité des ostéoclastes est régulée par l&#39;activateur du récepteur du facteur nucléaire-κ B ligand (RANKL), qui est produit par les ostéoblastes. Ostéoprotégérine (OPG), un récepteur leurre pour RANKL est également produite par les ostéoblastes 3 ​​Dans les modèles animaux in vivo qui impliquent la surexpression systémique de sRANKL, ou la suppression d&#39;OPG sont très précieux dans la recherche de l&#39;ostéoporose.; Cependant, ces méthodes nécessitent la génération de souris transgéniques à 4,5. Ici, une nouvelle alternativeméthode de surexpression sRANKL pour l&#39;étude des troubles musculo-squelettiques liés est décrite. Plus précisément, minicercles (MC) technologie de l&#39;ADN et les méthodes de livraison hydrodynamiques ont été utilisés pour réaliser le transfert de gènes in vivo de sRANKL et surexpriment la souris sRANKL systémique 6. Cette méthode est également complémentaire à d&#39;autres modèles in vivo de l&#39;ostéoporose, tels que la modulation hormonale des ostéoclastes suivants ovariectomie 7 et l&#39;intervention diététique par le régime alimentaire pauvre en calcium 8. Ces modèles sont très utiles pour étudier les différents aspects des troubles musculo-squelettiques liés-mais ils ont besoin d&#39;interventions chirurgicales et peuvent prendre jusqu&#39;à plusieurs mois, à un coût important 9. Ovariectomisées (OVX) modèle de rongeur est un modèle animal expérimental où l&#39;enlèvement des ovaires conduit à une déficience en oestrogènes mimant ainsi l&#39;ostéoporose post-ménopausique humaine 10. Human ostéoporose post-ménopausique, une condition où l&#39;oestrogène Deficirence conduit à un risque accru de fractures osseuses et l&#39;ostéoporose affecte environ huit millions de femmes aux États-Unis seulement. Bien que le modèle OVX est utile pour l&#39;ostéoporose post-ménopausique il offre des avantages limités à l&#39;étude de l&#39;ostéoporose en général. L&#39;œstrogène inhibe la perte osseuse, en induisant des ostéoclastes et en inhibant l&#39;apoptose des ostéoblastes, donc en l&#39;absence d&#39;une augmentation de l&#39;activité des ostéoclastes est observée 10 à 12. Un rapport RANKL-OPG déséquilibre qui favorise la résorption osseuse est également observée 13. Cependant, la carence en oestrogène in vivo est également accompagnée par des niveaux de transformation β du facteur de croissance (TGF β d&#39;), l&#39;augmentation de l&#39;interleukine-7 (IL-7) et le TNF, IL-1 et IL-6 14,15 diminué. Comme ces cytokines ont connu des fonctions modulatrices du remodelage osseux indépendante de la voie de RANKL, il n&#39;est pas possible d&#39;attribuer toute activation des ostéoclastes uniquement à l&#39;axe RANKL-RANK. Le modèle décrit dans ce document permet aux chercheurs d&#39;étudier en vivo axe RANKL-RANK dans ostéoclastogénèse et la perte osseuse sans cytokines pro-inflammatoires par rapport aux modèles de rongeurs OVX. En outre, les techniques in vitro ostéoclastogenèse sont des outils essentiels pour étudier l&#39;activation des ostéoclastes pour des traitements thérapeutiques potentielles des maladies musculo-squelettiques. Des études antérieures ont également montré que la culture de macrophages de souris dérivées de la moelle osseuse (BMMS) avec des macrophages de souris colony-stimulating factor (M-CSF) et de la souris sRANKL peut conduire à la différenciation des ostéoclastes 3,16,17. Ici, les protocoles pour générer des cellules ostéoclastes multinucléés analogue à partir de moelle osseuse de souris, ainsi que de cellules mononucléaires périphériques humaines du sang périphérique (PBMC) in vitro 18 sont décrits. Les tests cellulaires nécessaires pour définir un ostéoclastes différenciation terminale et entièrement fonctionnel maturité sont aussi brièvement décrits. Ces techniques in vitro complètent la nouvelle approche in vivo et, ensemble, servent de poutils d&#39;investigation owerful pour étudier la différenciation des ostéoclastes et activation. L&#39;utilisation de ces systèmes, les scientifiques sont capables de générer des ostéoclastes in vivo et in vitro et de définir les stimuli et les signaux nécessaires à leur prolifération et l&#39;activation ainsi que de tester l&#39;efficacité des inhibiteurs pharmacologiques et biologiques.

<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.

Ici, une technique de transfert de gène selon l&#39;invention pour la différenciation des ostéoclastes in vivo et des protocoles de culture de cellules pour la différenciation des cellules précurseurs des ostéoclastes in vitro en tant que méthode pour étudier les effets des cytokines sur l&#39;ostéoclastogenèse sont décrits. Sur la figure 1, les résultats représentatifs de transfert de gène réussie de GFP et de la souris sRANKL MC chez la souris sont présentées. Dans <...

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; Technique Gene Transfer et<em&gt; In vitro</em&gt; Essais cellule sur la base de l&#39;étude de la perte osseuse dans les troubles musculo-squelettiques

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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Universidad de Cartagena UDEC

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 vues.  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. 

Vidéo: 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.

Traitement de la toxicomanie et<em&gt; In Vivo</em&gt; Imagerie des ostéoblastes-ostéoclastes Interactions dans un modèle Medaka poisson Ostéoporose

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

Recherche

Biologie du développement

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.

Modèles de métastases osseuses

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

Médecine

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.

Séquentiel<em&gt; In vivo</em&gt; Imagerie des cellules souches / progénitrices ostéogéniques lors de la réparation de la fracture

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.

Combiné<em&gt; In vivo</em&gt; Optique et μCT imagerie pour surveiller l&#39;infection, l&#39;inflammation, et Bone Anatomy dans une infection orthopédique Implant souris

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.

des applications de<em&gt; In Vivo</em&gt; Tests fonctionnels du Rat jambier antérieur pour l&#39;évaluation de l&#39;ingénierie tissulaire Skeletal Muscle Repair

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

Bio-ingénierie

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 Fenêtre Chambre Modèle pour<em&gt; In Vivo</em&gt; Suivi des cellules dans la moelle osseuse murine

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

Immunologie et infection

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.

Semi-automatisées longitudinale axée sur les Microcomputed tomographie Quantitative analyse structurale d’un modèle de Rat nu Fracture vertébrale liée à l’ostéoporose

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

A Lab-On-A-Chip Platform for Stimulating Osteocyte Mechanotransduction and Analyzing Functional Outcomes of Bone Remodeling

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical environment. During this process, mechanosensitive osteocytes regulate the opposing responses between the catabolic osteoclasts and anabolic osteoblasts. To better understand the highly intricate signaling pathways that regulate this process, our lab has developed a foundationary lab-on-a-chip (LOC) platform for analyzing functional outcomes (formation and resorption) of bone remodeling within a small scale system. As bone remodeling is a lengthy process that occurs on the order of weeks to months, we developed long-term cell culturing protocols within the system. Osteoblasts and osteoclasts were grown on functional activity substrates within the LOC and maintained for up to seven weeks. Afterward, chips were disassembled to allow for the quantification of bone formation and resorption. Additionally, we have designed a 3D printed mechanical loading device that pairs with the LOC platform and can be used to induce osteocyte mechanotransduction by deforming the cellular matrix. We have optimized cell culturing protocols for osteocytes, osteoblasts, and osteoclasts within the LOC platform and have addressed concerns of sterility and cytotoxicity. Here, we present the protocols for fabricating and sterilizing the LOC, seeding cells on functional substrates, inducing mechanical load, and disassembling the LOC to quantify endpoint results. We believe that these techniques lay the groundwork for developing a true organ-on-a-chip for bone remodeling.

Here, we present protocols for analyzing bone remodeling within a lab-on-a-chip platform. A 3D printed mechanical loading device can be paired with the platform to induce osteocyte mechanostransduction by deforming the cellular matrix. The platform can also be used to quantify bone remodeling functional outcomes from osteoclasts and osteoblasts (resorption/formation).

Une plate-forme Lab-On-A-Chip pour stimuler la mécanotransduction ostéocyte et analyser les résultats fonctionnels du remodelage des os

Bone remodeling is a tightly regulated process that is required for skeletal growth and repair as well as adapting to changes in the mechanical ...

<meta charset="utf8"></head><body>Cellules dérivées d’ostéo-organoïdes pour une GCSH efficace chez des souris irradiées

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

<meta charset="utf8"></head><body>Approche ostéo-organoïde in vivo pour la récolte de cellules souches/progénitrices hématopoïétiques thérapeutiques

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Approche ostéo-organoïde in vivo pour la récolte de cellules souches/progénitrices hématopoïétiques thérapeutiques

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

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Semi-automatisées longitudinale axée sur les Microcomputed tomographie Quantitative analyse structurale d’un modèle de Rat nu Fracture vertébrale liée à l’ostéoporose

Une plate-forme Lab-On-A-Chip pour stimuler la mécanotransduction ostéocyte et analyser les résultats fonctionnels du remodelage des os

Approche ostéo-organoïde in vivo pour la récolte de cellules souches/progénitrices hématopoïétiques thérapeutiques

Approche ostéo-organoïde in vivo pour la récolte de cellules souches/progénitrices hématopoïétiques thérapeutiques