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. Hydrodynamische Lieferung der sRANKL MC DNA <ol><li> Hydrodynamische Lieferung über Mausschwanzvene <ol><li> Wiegen Sie die Maus vor der Schwanzveneninjektion. Verdünnen sRANKL oder grün fluoreszierende Protein (GFP) MC in Ringer-Lösung (pre-warmen bei 37 ° C) in einem Gesamtvolumen von ca. 10% der Mauskörpergewicht. </li><li> Wärmen Sie die Maus in einem Käfig für 10 Minuten vor der Injektion, um die Blutgefäße erweitern und machen seitlichen Venen (LVs) sichtbar. Überwachen Sie die Maus sorgfältig zu Dehydrierung und Hyperthermie zu vermeiden. </li><li> Sobald die LVs sind erweitert und sichtbar, übertragen Sie die Maus auf den Restrainer und den Stecker weit genug in den Lauf, um die Bewegung zurückzuhalten. Überwachen Sie die Maus für die normale Aktivität wie Atmung. Stellen Sie den Stecker des Restrainer, wenn nötig. </li><li> Desinfizieren Sie die Injektionsbereich 3/4 von der Spitze des Schwanzes mit einem Alkoholtupfer. Verwenden Sie eine 27 bis 30 G-Nadel für die Mausschwanzveneninjektion. Halten Sie den Schwanz mit einer Hand fest und legen Sie die Nadel inin die Schwanzvene. Üben Sie Druck auf die Spritze und füllen Sie die Injektion innerhalb von 5-7 Sekunden. </li><li> Entfernen Sie die Nadel aus der Schwanzvene und Druck mit einem Wattebausch auf die Injektionsstelle, um Blutungen zu stoppen. Überwachen Sie die Maus für 15-30 min, übertragen Sie dann die Maus wieder auf, sobald das Vivarium Atemfrequenz wird auf seiner normalen Geschwindigkeit reduziert. </li></ol></li><li> Bestätigung der Maus Systemische sRANKL Expression und Quantifizierung der Maus Serum TRACP5b Beitrag Gentransfer <ol><li> Wärmen Sie die Maus in einem Käfig für 5-10 min, um die Blutgefäße erweitern und machen LVs sichtbar. Überwachen Sie die Maus sorgfältig zu Dehydrierung und Hyperthermie zu vermeiden. </li><li> Machen Sie einen kleinen Schnitt mit einem Messer an der Maus den Schwanz an einem Ort, ein Viertel von der Spitze des Schwanzes. </li><li> Sammeln Sie etwa 100 ul Blut im Serum Trennung Röhren. </li><li> Serumtrennröhrchen bei Raumtemperatur inkubiert für 30 min. </li><li> Zentrifugieren Sie die Proben bei 10.000 xg für 5 min und sammeln Serum. Store Serum -80 ° C zur weiteren Analyse. </li><li> Führen sRANKL ELISA und Mausserum TRACP5b Assay auf Serum-Proben unter Verwendung eines kommerziell erhältlichen Kits. </li></ol></li></ol> 2. Osteoklasten in vitro Generierung von Maus BMM <ol><li> Isolierung und Kultivierung von BMM <ol><li> Die Isolierung des Schien-und Oberschenkelknochen: Euthanize den Spender-Maus mit CO 2, und desinfizieren Sie die Beine mit 70% Ethanol. Präparieren Sie vom Schambein zu dem Fersenbein, um die Oberschenkel und Schienbein Knochen entfernen intakt. </li><li> Entfernen Sie die Haut-und Muskel sorgfältig, ohne die Knochen. Platzieren verarbeitet Femur und Tibia Knochen in eine Petrischale mit phosphatgepufferter Salzlösung (PBS). </li><li> Entfernung von Knochenmark: Schneiden Sie die Spitze auf der einen Seite der Oberschenkel und Schienbein Knochen und spülen, die Knochenmark in einen 50-ml-Tube mit einer 1 ml-Spritze mit einer 25 G-Nadel, mit α-MEM mit 1% Penicillin / Streptomycin belastet (Osteoklasten Kultur Medium / OCM). </li><li&gt; Prozessknochenmark: Mix und übertragen Knochenmark-Suspension auf einen neuen 50-ml-Tube, indem es durch einen 70 um Nylon Zellsieb. Zentrifugation der Zellen bei 300 × g und resuspendieren in α-MEM, das 1% Penicillin / Streptomycin und 10% FBS (Osteoklasten-Kulturmedium / OCM (+)). </li></ol></li><li> Kultur von Makrophagen <ol><li> Zählen von Zellen mit einer Zählkammer oder automatische Zellzähler. </li><li> Platte 1 x 10 6 Zellen pro Well einer 6-Well-Platte oder 3 × 10 5 Zellen auf Glasdeckgläschen (5 mm) oder Dentinscheiben in 96-Well-Platte gelegt und bei 37 ° C </li><li> Saugen Sie alle Medien, die nicht-adhärenten Zellen und Inkubation Zellen bei 37 ° C mit frischem OCM (+) mit 25 ng / ml Maus M-CSF für 48 Stunden. </li><li> Saugen Sie alle Medien Zellen und Inkubation bei 37 ° C mit frischem OCM (+)-Medien mit 25 ng / ml Maus M-CSF und 30 ng / ml Maus sRANKL Osteoklastogenese zu initiieren. Tanken Medien alle 3 Tage nach Bedarf. </li><li> Zellen wachsenca. 6-8 Tage, um das riesige Mehrkernzellen, die Resorption Knochen zu bilden. Sobald kernige Zellen erscheinen, führen TRAP (Tartrat-resistente saure Phosphatase)-Färbung mit einem kommerziell erhältlichen Kit, und als voll ausgereift, führen Funktionstests. Visualisieren F-Aktin-Ringe auf Deckgläsern mit Phalloidin-Färbung und Bild die Zellen angebracht, um Scheiben durch Rasterelektronenmikroskopie (SEM), wie zuvor beschrieben 19 Dentin. </li></ol></li></ol> 3. Osteoklasten in vitro-Erzeugung aus Menschen PBMCs <ol><li> Isolierung von humanen PBMCs <ol><li> 10 ml vorgewärmten Histopaque-1077 in ein 50-ml-Tube. </li><li> Leere Leukozytenfilter aus Blutbank mit sterilem PBS erhalten in einen 50-ml-Tube (1:1-Verhältnis von Blut mit PBS). Schicht die verdünnte Blut über die Histopaque Lösung sehr langsam, dafür sorgen, dass das Blut nicht mit dem Histopaque mischen. </li><li> Zentrifuge Proben bei 1000 g für 20 min bei 18 ° C. Folgening Zentrifugation sorgfältig isolieren weiß Buffy-Coat-Schicht, die die weißen Blutkörperchen und übertragen auf ein neues 50-ml-Tube. </li><li> Zellen mit PBS verdünnen und Zentrifuge wieder bei 650 g für 5 min, um die Zellen zu sammeln. </li><li> Überstand entfernen und resuspendieren Zellen in OCM (-) Medien und zählen Zellen mit Zellzähler. </li></ol></li><li> Plating und Kultivierung PBMCs <ol><li> Resuspendieren der Zellen in OCM (+)-Medium und der Platte 8 x 10 6 Zellen / ml pro Vertiefung einer 6-Well-Platte oder 1 x 10 6 Zellen / ml pro Vertiefung in 96-Well-Platte. Platten Zellen auf Glasdeckgläschen (5 mm), die für Immunfluoreszenz-Bildgebung als auch auf Knochenscheiben für Knochenresorption Assays, wie zuvor beschrieben 20 erforderlich. </li><li> Änderung Kulturmedien, Auffüllen der Zellen mit menschlichen M-CSF alle 2-3 Tage oder nach Bedarf. Dann Differenzierungsschritt am Tag 5 gehen durch Zugabe von 30 ng / ml humanes sRANKL zusammen mit 25 ng / ml menschlichen M-CSF in OCM (+)-Medium zu initiieren osteoclastogenesist. </li><li> Wachsen Zellen für ca. 14 bis 21 Tage, um das riesige Mehrkern Zellen zu bilden. Sobald kernige Zellen angezeigt werden, können diese Zellen dann für TRAP beschriftet werden, und als voll ausgereift, können funktionelle Assays durchgeführt werden. F-Actin-Ringe können auf Deckgläsern mit Phalloidin-Färbung sichtbar gemacht werden. Die Morphologie der Zellen in Zellen angebracht, um Scheiben von SEM Dentin untersucht werden. </li></ol></li></ol>

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The authors have nothing to disclose.

Erkrankungen des Bewegungsapparats sind die führenden Ursachen von Morbidität und Behinderung und werden von über 150 Krankheiten und Syndrome besteht; Auswirkungen auf heute etwa 90 Millionen Amerikaner. Gelenkentzündungen und Knochenzerstörung wiegen Merkmale Erkrankungen des Bewegungsapparats, einschließlich Arthritis und Osteoporose. Osteoporose ist eine Erkrankung, die Knochen schwächt Integrität, die häufig Frakturen der Knochen führt. Arthritis ist eine chronische, schwächende Erkrankung, die durch Ent...

Muskel-Skelett-Erkrankungen betreffen Millionen von Menschen in den Vereinigten Staaten und Gegenwart schwerwiegende Folgen für die nationalen und lokalen Gesundheitssysteme ein. Diese Erkrankungen sind durch den Verlust von Knochen-und Gelenkfunktion, die umfangreiche Behandlung und lange Erholungsphasen erfordern gekennzeichnet. Üblicherweise wird eine relative Erhöhung der Anzahl und / oder Aktivität von Osteoklasten, spezialisierte Zellen zu Knochenresorption, Osteoporose und Arthritis beobachtet 2. Unter physiologischen Bedingungen ist die Anzahl und Aktivität von Osteoklasten durch Rezeptor-Aktivator-von Kernfaktor κ-B-Liganden (RANKL), die von Osteoblasten produziert wird geregelt. Osteoprotegerin (OPG), wird ein Köder Rezeptor für RANKL auch durch Osteoblasten 3 erzeugten In-vivo-Tiermodelle, die systemische Überexpression von sRANKL oder Deletion von OPG einzubeziehen sind sehr wertvoll bei Osteoporose-Forschung. Allerdings erfordern diese Verfahren die Herstellung von transgenen Mäusen 4,5. Hier wird eine neuartige AlternativeVerfahren überexprimieren sRANKL für die Untersuchung von Muskel-Skelett-bedingten Erkrankungen beschrieben. Insbesondere Minicircle (MC) DNA-Technologie und hydrodynamischen Versandmethoden wurden verwendet, um Gen-Transfer von sRANKL in vivo zu erreichen und überexprimieren Maus sRANKL systemisch 6. Diese Methode ist auch eine Ergänzung zu anderen in-vivo-Modelle von Osteoporose, wie hormonelle Modulation der Osteoklasten folgenden ovariectomy 7 und diätetische Intervention durch kalziumarme Ernährung 8. Diese Modelle sind sehr nützlich, um verschiedene Aspekte des Bewegungsbedingten Störungen aber sie erfordern chirurgische Verfahren und kann bis zu mehreren Monaten, was mit erheblichen Kosten 9 studieren. Ovariektomierte (OVX) Nagetier-Modell ist ein Modell, bei dem Versuchstier Entfernung der Eierstöcke führt zu Östrogenmangel, wodurch menschliche postmenopausalen Osteoporose 10 imitiert. Menschen postmenopausalen Osteoporose, eine Erkrankung, bei Östrogen deficirenz führt zu einem erhöhten Risiko von Knochenbrüchen und Osteoporose rund acht Millionen Frauen allein in den Vereinigten Staaten. Obwohl die OVX Modell ist nützlich für postmenopausalen Osteoporose bietet begrenzte Vorteile bei der Untersuchung von Osteoporose im Allgemeinen. Östrogen unterdrückt Knochenverlust, durch Induktion von Osteoklasten und Osteoblasten Hemmung der Apoptose, also in seiner Abwesenheit eine erhöhte Aktivität der Osteoklasten beobachtet 10-12. Ein RANKL-OPG-Verhältnis Ungleichgewicht, das die Knochenresorption begünstigt wird auch beobachtet, 13. Jedoch Östrogenmangel in vivo auch begleitet von verringerten Niveaus des transformierenden Wachstumsfaktor β (TGF β), erhöhte Interleukin-7 (IL-7) und TNF, IL-1 und IL-6 14,15. Da diese Zytokine Knochenumbau modulatorische Funktionen der RANKL Weg unabhängig bekannt ist, ist es unmöglich, eine Osteoklastenaktivierung nur auf die RANKL-RANK-Achse zuzuordnen. Die in diesem Dokument beschriebene Modell ermöglicht es Forschern, in viv studierenRANKL-RANK-o-Achse in Osteoklastogenese und Knochenverlust ohne pro-inflammatorischen Zytokinen im Vergleich zu OVX Nagetiermodellen. Zusätzlich kann in vitro Osteoklastogenese Techniken sind wesentliche Instrumente zur Aktivierung der Osteoklasten für mögliche therapeutische Behandlungen von Muskel-Skelett-Erkrankungen zu studieren. Frühere Studien haben auch gezeigt, dass die Kultivierung Knochenmark der Maus Makrophagen (BMM) mit Maus-Makrophagen-Kolonie-stimulierenden Faktor (M-CSF) und Maus sRANKL können, um die Differenzierung von Osteoklasten 3,16,17 führen. Hier werden die Protokolle kernige Osteoklasten-ähnliche Zellen aus Maus-Knochenmark sowie aus menschlichen peripheren mononukleären Blutzellen (PBMCs) in vitro zu erzeugen 18 beschrieben. Die zellbasierte Assays erforderlich, um eine reife terminal differenzierten und voll funktions Osteoklasten definieren ebenfalls kurz beschrieben. Diese in-vitro-Techniken ergänzen den Roman in vivo Ansatz zusammen und dienen als powerful Untersuchungswerkzeuge, um die Differenzierung von Osteoklasten und Aktivierung zu studieren. Mit Hilfe dieser Systeme sind die Wissenschaftler in der Lage, die Osteoklasten in vivo und in vitro zu erzeugen und definieren Sie die Reize und Signale für ihre Verbreitung und Aktivierung erforderlich sowie testen die Wirksamkeit von pharmakologischen und biologischen Inhibitoren.

<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.

Hier wird ein neuartiges Gentransfertechnik zur Differenzierung von Osteoklasten in vivo Zellkulturprotokolle zur Differenzierung in Osteoklasten-Vorläuferzellen in vitro als ein Verfahren, um die Wirkungen von Cytokinen auf die Entstehung von Osteoclasten zu studieren beschrieben. In Fig. 1 sind die repräsentativen Ergebnisse erfolgreicher Gentransfer von GFP und Maus sRANKL MC in Mäusen gezeigt. In Abbildung 2 sind die repräsentativen Bilder von Knochenmark der 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; Gentransfer-Technik und<em&gt; In-vitro-</em&gt; Zellbasierte Assays für das Studium der Knochenverlust in Skeletterkrankungen

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

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

Research

JoVE Journal

Medicine

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

11.6K Aufrufe.  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. 

Serie: 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.

Longitudinal Evaluation of Mouse Hind Limb Knochenverlust nach Rückenmarksverletzungen mit 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 ...

Forschung

Medizin

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.

Medikamentöse Behandlung und<em&gt; In Vivo</em&gt; Imaging von Osteoblasten-Osteoklasten-Wechselwirkungen in Medaka-Fisch Osteoporose Modell

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

Entwicklungsbiologie

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.

Models von Knochenmetastasen

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.

Modellierung von primären Knochentumoren und Knochenmetastasen mit Implantation eines soliden Tumortransplantats in den Knochen

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

Krebsforschung

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.

Sequentiell<em&gt; In-vivo-</em&gt; Imaging von osteogenen Stammzellen / Vorläuferzellen während Fracture Repair

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.

Visualisierung von Angiogenese durch Multiphoton Mikroskopie In Vivo in gentechnisch veränderten 3D-PLGA/nHAp Gerüst für Calvarial kritische Knochen defekt Reparatur

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

Biotechnik

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.

Femur Fensterkammer Modell für<em&gt; In Vivo</em&gt; Zelltracking im Knochenmark der Maus

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

Immunologie und Infektion

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.

Parallelisierter längs Microcomputed Tomographie-basierten quantitativen Strukturanalyse von einem nackten Rattenmodell Osteoporose bedingte Wirbelfraktur

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>Osteo-Organoid-abgeleitete Zellen für eine effektive HSZT bei bestrahlten Mäusen

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>In-vivo-Osteo-Organoid-Ansatz zur Gewinnung therapeutischer hämatopoetischer Stamm-/Vorläuferzellen

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.

In-vivo-Osteo-Organoid-Ansatz zur Gewinnung therapeutischer hämatopoetischer Stamm-/Vorläuferzellen

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

A Novel

Zusammenfassung

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Longitudinal Evaluation of Mouse Hind Limb Knochenverlust nach Rückenmarksverletzungen mit Novel, In vivo Methodology

Medikamentöse Behandlung und

Models von Knochenmetastasen

Modellierung von primären Knochentumoren und Knochenmetastasen mit Implantation eines soliden Tumortransplantats in den Knochen

Sequentiell

Visualisierung von Angiogenese durch Multiphoton Mikroskopie In Vivo in gentechnisch veränderten 3D-PLGA/nHAp Gerüst für Calvarial kritische Knochen defekt Reparatur

Femur Fensterkammer Modell für

Parallelisierter längs Microcomputed Tomographie-basierten quantitativen Strukturanalyse von einem nackten Rattenmodell Osteoporose bedingte Wirbelfraktur

In-vivo-Osteo-Organoid-Ansatz zur Gewinnung therapeutischer hämatopoetischer Stamm-/Vorläuferzellen

In-vivo-Osteo-Organoid-Ansatz zur Gewinnung therapeutischer hämatopoetischer Stamm-/Vorläuferzellen