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. Consegna idrodinamica di sRANKL MC DNA <ol><li> Consegna idrodinamica via Tail Vena del mouse <ol><li> Pesare il mouse prima dell&#39;iniezione coda vena. Diluire sRANKL o proteina fluorescente verde (GFP) MC in soluzione di Ringer (pre-riscaldare a 37 ° C) in un volume totale di circa 10% del peso corporeo del topo. </li><li> Riscaldare il topo in una gabbia per 10 min prima dell&#39;iniezione per dilatare i vasi sanguigni e rendere vene laterali (LV) visibili. Monitorare con attenzione il mouse per evitare la disidratazione e ipertermia. </li><li> Non appena i VL sono dilatati e visibili, trasferire il mouse per il limitatore e inserire la spina abbastanza lontano nel barile per frenare il movimento. Monitorare il mouse per il normale attività come la respirazione. Regolare la spina del dispositivo di immobilizzazione, se necessario. </li><li> Disinfettare la zona di iniezione 3/4 dalla punta della coda con un batuffolo imbevuto di alcool. Utilizzare un ago G 27-30 per l&#39;iniezione coda vena mouse. Tenere la coda saldamente con una mano e inserire l&#39;ago inla vena della coda. Applicare una pressione sulla siringa e completare l&#39;iniezione entro 5-7 sec. </li><li> Rimuovere l&#39;ago dalla vena della coda e applicare una pressione con un batuffolo di cotone sul sito di iniezione per fermare l&#39;emorragia. Monitorare il mouse per 15-30 minuti, quindi trasferire il mouse fino al vivaio una volta che il tasso di respirazione è ridotta al suo tasso normale. </li></ol></li><li> Conferma di espressione sistemica mouse sRANKL e quantificazione dei mouse Serum TRACP5b post Gene Transfer <ol><li> Riscaldare il topo in una gabbia per 5-10 minuti, al fine di dilatare i vasi sanguigni e rendere visibile LV. Monitorare con attenzione il mouse per evitare la disidratazione e ipertermia. </li><li> Fare una piccola incisione con una lama di coda del topo in un sito un quarto dalla punta della coda. </li><li> Raccogliere circa 100 ml di sangue in provette di siero di separazione. </li><li> Incubare le provette di siero di separazione a temperatura ambiente per 30 minuti. </li><li> Centrifugare i campioni a 10.000 xg per 5 minuti e raccogliere il siero. Store siero in -80 ° C per ulteriori analisi. </li><li> Eseguire sRANKL ELISA e mouse dosaggio TRACP5b siero su campioni di siero utilizzando un kit commerciale disponibile. </li></ol></li></ol> 2. In vitro Osteoclasta generazione di mouse BMMs <ol><li> Isolamento e Coltura di BMMs <ol><li> Isolamento della tibia e femore ossa: Euthanize il mouse donatore con CO 2, e disinfettare le gambe con il 70% di etanolo. Sezionare dal pube all&#39;osso calcagno, per rimuovere femore e tibia ossa intatte. </li><li> Rimuovere la pelle ei muscoli attenzione senza danneggiare l&#39;osso. Posizionare femore e tibia trasformati in una capsula di Petri contenente tampone fosfato salino (PBS). </li><li> Rimozione di midollo osseo: Tagliare la punta su un lato del femore e della tibia ossa e lavare il midollo osseo in una provetta da 50 ml con una siringa da 1 ml con un ago G 25, caricato con α-MEM contenente 1% di penicillina / streptomicina (osteoclasti cultura medio / OCM). </li><li&gt; Processo midollo osseo: Mescolare e trasferire sospensione midollo osseo in un tubo da 50 ml facendola passare attraverso un filtro 70 micron cella nylon. Centrifugare le cellule a 300 xg e risospendere in α-MEM contenente 1% di penicillina / streptomicina e 10% FBS (Osteoclasta colturale / OCM (+)). </li></ol></li><li> Cultura dei macrofagi <ol><li> Contare le cellule utilizzando un emocitometro o contatore di cellule automatico. </li><li> Piastra 1 x 10 6 cellule per bene per un 6-pozzetti o 3 x 10 5 cellule per vetrino (5 mm) o fette di dentina collocati in piastra a 96 pozzetti ed incubare a 37 ° C. </li><li> Aspirare tutti i supporti contenenti cellule non aderenti e Incubare le cellule a 37 ° C con OCM fresco (+) contenente topo 25 ng / ml M-CSF per 48 ore. </li><li> Aspirare tutti i media e incubare le cellule a 37 ° C con nuova OCM (+) supporti contenenti il ​​mouse 25 ng / ml M-CSF e 30 ng / ml il mouse sRANKL per avviare osteoclastogenesis. Rifornire i media ogni 3 giorni come richiesto. </li><li> Crescere celluleper circa 6-8 giorni per formare celle multi-nucleari giganti in grado di riassorbimento osseo. Non appena compaiono le cellule multinucleate, eseguire TRAP (fosfatasi acida tartrato resistente) colorazione utilizzando un kit commerciale disponibile, e quando è completamente maturato, eseguire test funzionali. Visualizza anelli di F-actina su vetrini con macchia phalloidin e immagine le cellule attaccate alla dentina fette da microscopia elettronica a scansione (SEM), come descritto in precedenza 19. </li></ol></li></ol> 3. In vitro Osteoclasta generazione da PBMC umano <ol><li> Isolamento di PBMC umano <ol><li> Aggiungere 10 ml di pre-riscaldato Histopaque-1077 in una provetta da 50 ml. </li><li> Filtro leucociti vuoto ottenuto dalla banca del sangue con PBS sterile in una provetta da 50 ml (rapporto 1:1 di sangue con PBS). Strato il sangue diluito su tutto la soluzione HISTOPAQUE molto lentamente, facendo in modo che il sangue non si mescola con il HISTOPAQUE. </li><li> Centrifugare i campioni a 1000 xg per 20 min a 18 ° C. Seguireing centrifugazione, isolare attentamente il buffy bianco strato di rivestimento contenente i globuli bianchi e le trasferisce in un nuovo tubo da 50 ml. </li><li> Diluire le cellule con PBS e centrifugare di nuovo a 650 xg per 5 minuti per raccogliere le cellule. </li><li> Rimuovere il surnatante e risospendere le cellule in OCM (-) i media e contare le cellule utilizzando contatore di cellule. </li></ol></li><li> Placcatura e Coltura PBMC <ol><li> Risospendere le cellule in OCM (+) Media e piastra 8 x 10 6 cellule / ml per pozzetto in una piastra da 6 pozzetti o 1 x 10 6 cellule / ml per pozzetto in piastre da 96 pozzetti. Cellule piastra su vetrini (5 mm) come richiesto per l&#39;imaging immunofluorescenza nonché sulle fette di osso adatti per saggi di riassorbimento osseo come precedentemente descritto 20. </li><li> Cambia terreni di coltura, rifornire le cellule umane con M-CSF ogni 2-3 giorni o secondo necessità. Quindi procedere alla differenziazione passo il giorno 5 aggiungendo 30 ng / ml di sRANKL umana con 25 ng / ml M-CSF umano nella OCM (+) mezzi di avviare osteoclastogenesè. </li><li> Crescere cellule per circa 14 a 21 giorni per formare cellule multi-nucleari giganti. Non appena compaiono le cellule multinucleate, queste cellule possono quindi essere etichettati per TRAP, e quando è completamente maturato, test funzionali possono essere eseguiti. Anelli di F-actina possono essere visualizzati su vetrini con macchia falloidina. La morfologia cellulare può essere esaminato in cellule attaccate alla dentina fette mediante SEM. </li></ol></li></ol>

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The authors have nothing to disclose.

Condizioni muscolo-scheletriche sono le cause principali di morbilità e di disabilità e sono composti da oltre 150 malattie e sindromi; che colpisce circa 90 milioni di americani oggi. L&#39;infiammazione articolare e la distruzione ossea sono caratteristiche predominanti di disturbi muscolo-scheletrici, tra cui l&#39;artrite e l&#39;osteoporosi. L&#39;osteoporosi è una condizione che indebolisce l&#39;integrità ossea, causando spesso fratture delle ossa. L&#39;artrite è una malattia cronica, debilitante malattia c...

Malattie muscolo-scheletriche colpiscono milioni di persone negli Stati Uniti e presentare gravi conseguenze per i sistemi sanitari nazionali e locali 1. Questi disturbi sono caratterizzati da perdita di osso e funzione articolare che richiedono un trattamento estensivo e lunghi periodi di recupero. Comunemente, un relativo aumento del numero e / o attività di osteoclasti, cellule specializzate a riassorbire osso, osteoporosi ed artrite si osserva 2. In condizioni fisiologiche il numero e l&#39;attività degli osteoclasti è regolata da attivatore del recettore del fattore nucleare κ-B ligando (RANKL), che è prodotto dagli osteoblasti. Osteoprotegerina (OPG), un recettore decoy per RANKL è anche prodotta dagli osteoblasti 3 In modelli animali in vivo che coinvolgono la sovraespressione sistemica di sRANKL, o la cancellazione di OPG sono molto prezioso nella ricerca sull&#39;osteoporosi.; Tuttavia, questi metodi richiedono la generazione di topi transgenici 4,5. Qui, una nuova alternativametodo di iperespressione sRANKL per lo studio dei disturbi muscoloscheletrici legati è descritto. In particolare, minicircle (MC) tecnologia del DNA e metodi di consegna idrodinamiche sono stati usati per conseguire trasferimento genico di sRANKL in vivo e iperespressione del mouse sRANKL sistemicamente 6. Questo metodo è anche complementare ad altri modelli in vivo di osteoporosi, come la modulazione ormonale degli osteoclasti seguenti ovariectomia 7 e intervento dietetico da basso contenuto di calcio dieta 8. Questi modelli sono molto utili per studiare diversi aspetti dei disturbi muscoloscheletrici legati tuttavia essi richiedono procedure chirurgiche e può richiedere fino a diversi mesi, ad un costo significativo 9. Ovariectomizzate (OVX) modello di roditore è un modello animale sperimentale in cui la rimozione delle ovaie porta alla mancanza di estrogeni mimando così postmenopausale umano 10. Umano post-menopausa osteoporosi, una condizione in cui gli estrogeni deficirenza porta ad un aumento del rischio di fratture ossee e l&#39;osteoporosi colpisce circa otto milioni di donne negli Stati Uniti da soli. Anche se il modello OVX è utile per l&#39;osteoporosi post-menopausale che offre vantaggi limitati a studiare l&#39;osteoporosi in generale. Estrogeni sopprime perdita ossea, inducendo osteoclasti e osteoblasti inibizione dell&#39;apoptosi, quindi in sua assenza un aumento dell&#39;attività degli osteoclasti si osserva 10-12. Un rapporto di squilibrio RANKL-OPG che favorisce riassorbimento osseo si osserva anche 13. Tuttavia, la carenza di estrogeni in vivo è accompagnato anche da una diminuzione dei livelli di fattore di crescita trasformante β (TGF β), aumento della interleuchina-7 (IL-7) e TNF, IL-1 e IL-6 14,15. Poiché queste citochine hanno conosciuto funzioni modulatori rimodellamento osseo indipendente dal percorso che RANKL, è impossibile attribuire qualsiasi osteoclasti esclusivamente all&#39;asse RANKL-RANK. Il modello descritto in questo documento consente ai ricercatori di studiare in vivo l&#39;asse RANKL-RANK in osteoclastogenesi e la perdita di osso senza citochine pro-infiammatorie rispetto ai modelli di roditori OVX. Inoltre, le tecniche osteoclastogenesi in vitro sono strumenti essenziali per studiare l&#39;attivazione degli osteoclasti per i potenziali trattamenti terapeutici delle malattie muscolo-scheletriche. Precedenti studi hanno inoltre dimostrato che la coltura di midollo osseo di topo macrofagi derivati ​​(BMMs) con il mouse macrofagi fattore stimolante le colonie (M-CSF) e il mouse sRANKL può portare a osteoclasti differenziazione 3,16,17. Qui sono descritti i protocolli per generare cellule osteoclasti come multinucleate da midollo osseo di topo e di cellule mononucleari del sangue periferico (PBMC) in vitro 18. I saggi cellulari necessari per definire un maturo osteoclasti terminalmente differenziate e pienamente funzionale sono anche descritte brevemente. Queste tecniche in vitro completano il romanzo di approccio vivo e insieme fungono da pstrumenti investigativi owerful per studiare la differenziazione degli osteoclasti e l&#39;attivazione. L&#39;utilizzo di questi sistemi, gli scienziati sono in grado di generare osteoclasti in vivo e in vitro e definire gli stimoli e segnali necessari per la proliferazione e l&#39;attivazione e testare l&#39;efficacia di inibitori farmacologici e biologici.

<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.

Qui, una tecnica di trasferimento nuovo gene per la differenziazione degli osteoclasti in vivo e protocolli di coltura cellulare per differenziare le cellule precursori in osteoclasti in vitro come metodo per studiare gli effetti di citochine sulla osteoclastogenesis sono descritti. In Figura 1, sono mostrati i risultati rappresentativi di successo trasferimento genico di GFP e mouse sRANKL MC nei topi. Nella figura 2, vengono mostrate le immagini rappresentative del m...

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; Tecnica Gene Transfer e<em&gt; In vitro</em&gt; test cellulari per lo studio della perdita ossea nei disturbi muscoloscheletrici

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 Views.  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

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

La valutazione longitudinale di perdita dell&#39;arto posteriore ossea del mouse dopo la lesione del midollo spinale tramite Novel, In vivo, Metodologia

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Ricerca

Medicina

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.

Il trattamento di droga e<em&gt; In Vivo</em&gt; Imaging di osteoblasti-osteoclasti interazioni in un modello di Medaka pesce osteoporosi

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

Biologia dello sviluppo

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.

Modelli di metastasi ossee

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

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Modellazione dei tumori ossei primari e delle metastasi ossee con impianto di innesto tumorale solido nell'osso

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Ricerca sul cancro

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.

Sequenziale<em&gt; In vivo</em&gt; Imaging di osteogeniche cellule staminali / progenitrici durante la frattura di riparazione

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

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

Visualizzando l'angiogenesi da microscopia multifotonica In Vivo in geneticamente 3D-PLGA/nHAp impalcatura per Calvarial riparazione di difetti ossei critici

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Bioingegneria

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.

Femore finestra del modello Camera per<em&gt; In Vivo</em&gt; Monitoraggio delle cellule nel midollo osseo murino

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

Immunologia e infezioni

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.

Semiautomatica longitudinale basati su Microcomputed tomografia quantitativa analisi strutturale di un modello di relazione con l'osteoporosi fratture vertebrali del ratto nudo

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>Cellule derivate da osteo-organoidi per HSCT efficaci nei topi irradiati

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>Approccio osteo-organoide in vivo per la raccolta di cellule staminali/progenitrici ematopoietiche terapeutiche

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.

Approccio osteo-organoide in vivo per la raccolta di cellule staminali/progenitrici ematopoietiche terapeutiche

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

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La valutazione longitudinale di perdita dell'arto posteriore ossea del mouse dopo la lesione del midollo spinale tramite Novel, In vivo, Metodologia

Il trattamento di droga e

Modelli di metastasi ossee

Modellazione dei tumori ossei primari e delle metastasi ossee con impianto di innesto tumorale solido nell'osso

Sequenziale

Visualizzando l'angiogenesi da microscopia multifotonica In Vivo in geneticamente 3D-PLGA/nHAp impalcatura per Calvarial riparazione di difetti ossei critici

Femore finestra del modello Camera per

Semiautomatica longitudinale basati su Microcomputed tomografia quantitativa analisi strutturale di un modello di relazione con l'osteoporosi fratture vertebrali del ratto nudo

Approccio osteo-organoide in vivo per la raccolta di cellule staminali/progenitrici ematopoietiche terapeutiche

Approccio osteo-organoide in vivo per la raccolta di cellule staminali/progenitrici ematopoietiche terapeutiche