Diese Arbeit wurde von R01-AR053645 aus NIAMS finanziert.

Alle Arbeiten wurden von der UCSF Institutional Animal Care und Use Committee genehmigt und entsprechen den nationalen Richtlinien. 1. Vorbereitung der Fixatoren vor der Operation <ol><li> Vor dem Erstellen stabilisierte Fraktur, braucht man, um das benutzerdefinierte Design-Fixateur externe Gerät zusammenzubauen. Dem individuell gestalteten externe Fixation Rahmen bestand aus zwei Aluminium-Ringe von drei Edelstahl-# 0/56 Gewindestangen, 8 # 2/56 Sechskantmuttern und 17 passende Befestigungsschrauben (Small Part Inc., Miami Lakes, FL 3) stabilisiert. </li><li> Zwei Aluminium-Ringe sind pro Fraktur benötigt. Um einen Ring montieren 4 # 2/56 Sechskantmuttern (kleiner Teil Inc., Miami Lakes, FL) und passende Schrauben in die entsprechenden Löcher, sollten sie bleiben locker. Dies wird der proximale Ring sein. An dem distalen Ring montieren 3 Schrauben und Muttern, nicht erneut nicht voll anziehen. </li></ol> 2. Anästhesie-, Bruch der Schöpfung, und Fixateur <ol><li> Mäuse (Alter, Geschlecht, Körper undGew. werden von den Kunden bestimmt) werden anästhesiert durch intraperitoneale Injektion von Ketamin 50mg/ml, 0,5 mg / ml Medetomidin (0,03 ml / Maus). </li><li> Bewerben Auge Schmiermittel für die Augen der Mäuse und setzen Sie ein Stück Klebeband über ihren Kopf, ihre Augen während des Verfahrens zu schützen. </li><li> Das Tier wird auf einem Heizkissen für den Rest des Verfahrens platziert. </li><li> Transfix die proximale und distale Metaphyse der linken Tibia mit vier 0,25 mm sterilen Insekten Stifte (Korrosionsschutzbinde: Fine Science Tools, Foster City, CA) durch Anbringen der Stifte zu einem Dremel-Werkzeug und Bohren sie durch den Knochen. </li><li> Pins sind senkrecht zu der Längsachse der Tibia, und 90 ° zueinander. Mindestens 10 mm zwischen dem proximalen und distalen Stiften. </li><li> Positionieren Sie den ersten Ring über den proximalen Stifte mit den Muttern nach innen, in Richtung der Pins. Sichern Sie die Stifte in den Ring hält das Schienbein zentral innerhalb der Alu-Ring positioniert. </li><li> Positionieren Sie den zweiten Ring unterhalb des diStal Stifte und sichern Sie die Stifte in den Ring mit den Sechskantmuttern wie zuvor. Fügen Sie die vierte Mutter-Bolzen Kombination mit dem distalen Fixateur. </li><li> Erstellen Sie die Querfraktur durch Drei-Punkt-Biegung mit einer entsprechenden Vorrichtung. Wir nutzen das System der Schwerkraft Bonnarens und Einhorn 4. Zeigen Sie mit der posterolateralen Oberfläche des Schienbeins in den Blöcken, heben Sie das Gewicht auf eine vorgegebene Höhe entlang der Fallarm-and-Drop das Gewicht, so dass es Kontakte die anterolaterale Seite des Schienbeins, um den stumpfen Bruch zu erzeugen. Die Höhe wird das Gewicht von Bedürfnissen sank um für jeden Stamm der Maus, um einen einzigen Bruch des Schienbeins ausschließlich gewährleisten ermittelt werden. </li><li> Fraktur wird durch Röntgenstrukturanalyse bestätigt. </li><li> Nach der Bestätigung der Fraktur, sind drei Edelstahl # 0/56 Gewindestangen an den Ringen befestigt. Ausgehend von dem distalen Ende nach der Stange und Einfädeln einer Mutter auf dem distalen Ring, um die Stange zu sichern. Dann legen Sie eine andere Mutter auf der Stange, wird der proximale Ring an der Oberseite des n sitzenut. Stellen Sie sicher, dass die beiden Ringe in gleichen Abständen stattfinden wird und nicht belasten die Stifte. Sichern Sie den proximalen Ring an den Stäben mit einer dritten Mutter. </li><li> Subkutane Injektion von Buprenorphin (1,0 mg / kg) für die Analgesie werden sofort nach der Operation und die Tiere für Schmerz und Buprenorphin gegebenen überwacht nach Bedarf. </li><li> Anästhesie wird durch die Gabe von Atipamezol umgekehrt, und Tiere werden überwacht, bis sie reagieren und ambulating sind. </li></ol> 3. Distraktionsosteogenese (siehe auch: 12,13) Um dieses Verfahren zu ändern, um Platz für Distraktionsosteogenese ist unkompliziert. Die Ringe werden in ihrer Position durch Gewindestangen gehalten, und durch Drehen der Muttern, mit denen die Stäbe an Ort und Stelle können die Ringe auseinander bewegt werden. <ol><li> Latenz: Für Standard-Distraktionsosteogenese mit diesem Modell die Fixateure werden auf der Tibia platziert und allein für eine Latenzzeit von 5 Tage Zeit. </li><li> Ablenkung: Ablenkung osteogenesis kann durch Drehen der Muttern an den Stabilisierungsstangen ¼ Umdrehung (0,25 mm) jeden Tag 7 Tage durchgeführt werden. </li><li> Reifung: knöcherne Überbrückung wird durch 10 Tage lang beobachtet und vollständige Konsolidierung wird durch 27 Tage nach Abschluss der Ablenkung beobachtet. </li></ol> 4. Schaffung einer kritischen Sized Defect Um eine kritische Größe Defekt verursachen die Fixateur externe werden angewendet, wie zuvor mit den folgenden Modifikationen. <ol><li> Vor Beginn des Verfahrens wird die Haut mit 70% Ethanol gereinigt. </li><li> Die Fixateure sind komplett vor dem Erstellen der Defekt appliziert. </li><li> Die Maus wird dann unter dem Präpariermikroskop Ort, um die mediale Tibia zu visualisieren, und einen Einschnitt (5-7 mm) mit einem # 10 Klinge auf einem Skalpell. </li><li> Der Muskel wird durchtrennt und sanft getrennt, um die Mitte der Diaphyse der Tibia freizulegen. </li><li> Ein 3 mm Segment des Knochens wird dann ab Mitte der Diaphyse mit einer Schere entfernt. Mit ter Punkt der Schere wird der Knochen langsam weg und schnitt Knochenfragmente werden mit einer Pinzette bei Bedarf entfernt werden. Es sollte mit den Spitzen der Schere getroffen werden, um zu vermeiden Verschieben der Tibia mit einer großen mechanischen Kraft. </li><li> Untersuchen die chirurgische Stelle um sicherzustellen, dass keine Knochenfragmente, da diese die Heilung zu fördern könnte. </li><li> Ersetzen Sie die Muskeln rund um die abgestumpften Enden des Schienbeins. </li><li> Schließen Sie den Schnitt auf der Haut mit 2-3 Fäden von 6-0 Polyamin Monofilament. </li><li> Die Betäubung wird umgekehrt und die Tiere überwacht Schmerzen und gegebenen Analgetika, wie zuvor beschrieben. </li></ol> 5. Repräsentative Ergebnisse Wenn sie richtig angewendet wird, bieten die Fixateur externe steifer Stabilität des geschlossenen Tibiafraktur mit ausgezeichneten Reduktion (Abb. 1, 2). In einigen Fällen nicht ausreichend Reduktion (offensichtlich ein großer Spalt zwischen Knochenenden oder mehrere Brüche auftreten (Abb. 3 </strong&gt;), Und diese Mäuse werden von der Analyse ausgeschlossen. Frakturen stabilisiert mit dieser Methode heilen in erster Linie über intramembranöse Ossifikation (Abb. 4). Im Gegensatz dazu, wenn der Bruch nicht stabilisiert ist eine große Knorpel Kallus wird in den Spalt der Fraktur (5) gebildet ist, und dies wird durch Knochen durch das Verfahren nach intramembranöse Ossifikation ersetzt. <img alt="1" src="/files/ftp_upload/3552/3552fig1.jpg" /> 1. Röntgenaufnahmen, welches eine externe Fixiervorrichtung verwendet, um Tibiafraktur stabilisieren. Röntgenbild nach der Fraktur zeigen einen gut ausgerichteten Knochensegmenten (Pfeilspitze) übernommen. <img alt="2" src="/files/ftp_upload/3552/3552fig2.jpg" /> 2. Bild von einer Maus nach dem Fixateur angewendet wurde. <img alt="Abbildung 3" src="/files/ftp_upload/3552/3552fig3.jpg" /> Abbildung 3. Röntgenaufnahme nach f genommenracture zeigt falsch ausgerichtet und fragmentierten Knochen Segmente (Pfeilspitze). <img alt="Abbildung 4" src="/files/ftp_upload/3552/3552fig4.jpg" /> Abbildung 4. Stabilisierte Fraktur heilt über intramembranöse Ossifikation. Trichromfärbung von stabilisierten Fraktur zeigt einige neue Knochen (b) bei Fraktur. Maßstab = 500 um. <img alt="Abbildung 5" src="/files/ftp_upload/3552/3552fig5.jpg" /> Abbildung 5. Non-stabilisierte Fraktur heilt über enchondralen Ossifikation. Trichromfärbung von nicht stabilisierten Fraktur zeigt Knorpel (c) und Knochen (b) bei Fraktur. Maßstab = 500 um.

<ol>
	<li>Ilizarov, G. A., Lediaev, V. I., Shitin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+course+of+compact+bone+reparative+regeneration+in+distraction+osteosynthesis+under+different+conditions+of+bone+fragment+fixation+(experimental+study).">The course of compact bone reparative regeneration in distraction osteosynthesis under different conditions of bone fragment fixation (experimental study).</a> Eksperimentalnaia Khirurgiia i Anesteziologiia. 14, 3-12 (1969).</li><li>Ilizarov, G. A., Deviatov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+elongation+of+the+leg.">Surgical elongation of the leg.</a> Ortopediia Travmatologiia i Protezirovanie. 32, (1971).</li><li>Thompson, Z., Miclau, T., Hu, D., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+intramembranous+ossification+during+fracture+healing.">A model for intramembranous ossification during fracture healing.</a> J. Orthop. Res. 20, 1091-1098 (2002).</li><li>Bonnarens, F., Einhorn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+standard+closed+fracture+in+laboratory+animal.">Production of a standard closed fracture in laboratory animal.</a> J. Orthop. Res. 2, 97-101 (1984).</li><li>Colnot, C., Thompson, Z., Miclau, T., Werb, Z., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+fracture+repair+in+the+absence+of+MMP9.">Altered fracture repair in the absence of MMP9.</a> Development. 130, 4123-4133 (2003).</li><li>Lange, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+IL-1beta+during+fracture+healing.">Action of IL-1beta during fracture healing.</a> J. Orthop. Res. 28, 778-784 (2010).</li><li>Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+for+CCR2+during+fracture+healing.">Multiple roles for CCR2 during fracture healing.</a> Dis. Model Mech. 3, 451-458 (2010).</li><li>Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+human+bone+morphogenetic+protein-7+enhances+fracture+healing+in+an+ischemic+environment.">Recombinant human bone morphogenetic protein-7 enhances fracture healing in an ischemic environment.</a> J. Orthop. Res. , (2009).</li><li>Yu, Y. Y., Lieu, S., Lu, C., Colnot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein+2+stimulates+endochondral+ossification+by+regulating+periosteal+cell+fate+during+bone+repair.">Bone morphogenetic protein 2 stimulates endochondral ossification by regulating periosteal cell fate during bone repair.</a> Bone. 47, 65-73 (2010).</li><li>Lu, C., Miclau, T., Hu, D., Marcucio, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia+leads+to+delayed+union+during+fracture+healing:+a+mouse+model.">Ischemia leads to delayed union during fracture healing: a mouse model.</a> J. Orthop. Res. 25, 51-61 (2007).</li><li>Miclau, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+delayed+stabilization+on+fracture+healing.">Effects of delayed stabilization on fracture healing.</a> J. Orthop. Res. 25, 1552-1558 (2007).</li><li>Tay, B. K., Le, A. X., Gould, S. E., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+and+molecular+analyses+of+distraction+osteogenesis+in+a+mouse+model.">Histochemical and molecular analyses of distraction osteogenesis in a mouse model.</a> Journal of Orthopaedic Research. 16, 636-642 (1998).</li><li>Choi, P., Ogilvie, C., Thompson, Z., Miclau, T., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+characterization+of+a+murine+non-union+model.">Cellular and molecular characterization of a murine non-union model.</a> J. Orthop. Res. 22, 1100-1107 (2004).</li><li>Buckwalter, J. A., Marsh, E. T., J, L., Heckman, J. D., Bucholz, R. 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Wir haben nichts zu offenbaren.

Bones von zwei unterschiedlichen Modalitäten je nach mechanischer Stabilität (: 14 bewertet in) zu heilen. Wenn links instabil einen großen Knorpel-Vorlage Formen im Frakturspalt, die durch Knochen ersetzt wird, um die beiden Enden des gebrochenen Knochens zu überbrücken. Proximal und distal der Pause bildet der Knochen direkt durch intramembranöse Ossifikation im Periost und Endost. Im Gegensatz dazu tritt bei stabilen Frakturen Heilung ausschließlich über intramembranöse Ossifikation 3. ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> 0,25 mm Pin Insekt </td><td> Fine Science Tools </td><td> 26000-25 </td><td> Verdunkelten eloxiert Stahl, 0,25 mm Stangendurchmesser, 4cm Länge </td></tr><tr><td> Edelstahl-Sechskantmutter </td><td> Kleinteile </td><td> # 2-56 </td><td> 1/8 &quot;Länge, 56 Fäden pro Zoll </td></tr><tr><td> Edelstahl-Sechskantmutter </td><td> Kleinteile </td><td> # 0-80 </td><td> 1/8 &quot;lang, 80 Fäden pro Zoll </td></tr><tr><td> Stainless Steel Maschinenschraube </td><td> Kleinteile </td><td> # 0-80 </td><td> 1/8 &quot;lang, 80 Fäden pro Zoll </td></tr><tr><td> Edelstahl-Maschinen-Schnitt Gewindestange </td><td> Kleinteile </td><td> # 0-80 </td><td> 6 &quot;Länge, 80 Fäden pro Zoll </td></tr><tr><td> 18-8 Stainless Steel Head Machine ScREW </td><td> McMaster-Carr </td><td></td><td> 2-56 Gewinde, 3/6 &quot;Länge </td></tr><tr><td> Externe Befestigungsvorrichtung </td><td> Maschinenhalle </td><td> Kundenspezifische </td><td></td></tr></tbody></table>

creating rigidly stabilized fractures for assessing intramembranous ossification, distraction osteogenesis, or healing of critical sized defects

Beurteilung Modi Skelettreparatur notwendig für die Entwicklung von Therapien, um klinisch verwendet werden, um Frakturen. Mechanische Stabilität spielt eine große Rolle bei der Heilung von Knochenverletzungen. Im schlimmsten Fall mechanische Instabilität kann zu verzögerten oder nicht gewerkschaftlich beim Menschen führen. Allerdings kann auch Bewegung regen den Heilungsprozess. Bei Frakturen, die Bewegung Knorpel Formen haben, um die Fraktur Knochenenden zu stabilisieren, und dies Knorpel allmählich durch Knochen durch Rekapitulation des Entwicklungsprozesses von enchondralen Ossifikation ersetzt. Im Gegensatz dazu, wenn ein Knochenbruch starr stabilisiert ist Knochen bildet direkt über intramembranöse Ossifikation. Klinisch sowohl enchondralen und intramembranöse Ossifikation gleichzeitig auftreten. Um effektiv zu replizieren diese Verfahren Ermittler mit einer Nadel in die Markhöhle des gebrochenen Knochens durch Bonnarens 4 beschrieben. Diese experimentelle Methode bietet eine hervorragende seitliche Stabilität und ermöglicht RotatioNAL Instabilität zu bestehen. Allerdings kann unser Verständnis der Mechanismen, die diese beiden unterschiedlichen Prozesse regulieren auch experimentell durch Isolieren jeder dieser Prozesse verbessert werden. Wir haben eine Stabilisierung Protokoll, das Rotations-und seitlichen Stabilisierung liefert entwickelt. In diesem Modell ist intramembranöse Ossifikation die einzige Art der Heilung, die beobachtet wird, und Heilung Parameter können zwischen verschiedenen Stämmen von Mäusen 5-7, nach dem Aufbringen von bioaktiven Molekülen 8,9, nach der Änderung physiologische Parameter der Heilung 10 verglichen werden, nach der Änderung der Menge oder der Zeitpunkt der Stabilisierung 11 nach Distraktionsosteogenese 12, nach der Erzeugung eines nicht-Einheit 13, oder nach dem Erstellen einer kritischen Größe Defekt. Hier zeigen wir, wie man die modifizierte Ilizarov Fixateur für das Studium Tibia Frakturheilung und Distraktionsosteogenese bei Mäusen anzuwenden.

Watch this Scientific Journal Video about Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects at JoVE.com

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Dieser Artikel beschreibt ein Verfahren zur Stabilisierung von Frakturen langer Röhrenknochen, die über die Anwendung der modifizierten Ilizarov Fixateur externe beruht<sup&gt; 3.1</sup&gt;. Nach Anwendung der Fixateur und Schaffung des Knochen-Verletzungen, Heilung beurteilt werden kann, können Distraktionsosteogenese durchgeführt werden, oder nicht gewerkschaftlich oder kritische Größe Defekt kann erstellt und verwendet werden, um therapeutische Interventionen zu studieren.

Erstellen Starr stabilisierte Frakturen zur Beurteilung intramembranöse Ossifikation, Distraktionsosteogenese, oder Heilung von kritischer Größe Mängel

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

creating-rigidly-stabilized-fractures-for-assessing-intramembranous

Research

JoVE Journal

Medicine

17.2K Aufrufe.  University of California, San Francisco. Dieser Artikel beschreibt ein Verfahren zur Stabilisierung von Frakturen langer Röhrenknochen, die über die Anwendung der modifizierten Ilizarov Fixateur externe beruht 3.1. Nach Anwendung der Fixateur und Schaffung des Knochen-Verletzungen, Heilung beurteilt werden kann, können Distraktionsosteogenese durchgeführt werden, oder nicht gewerkschaftlich oder kritische Größe Defekt kann erstellt und verwendet werden, um therapeutische Interventionen zu ...

Serie: Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. Adipose-derived stromal cells (ASCs) have proven to be an abundant source of multipotent stem cells capable of undergoing osteogenic, chondrogenic, adipogenic, and myogenic differentiation. Many studies have explored the osteogenic potential of these cells in vivo with the use of various scaffolding biomaterials for cellular delivery. It has been demonstrated that by utilizing an osteoconductive, hydroxyapatite-coated poly(lactic-co-glycolic acid) (HA-PLGA) scaffold seeded with ASCs, a critical-sized calvarial defect, a defect that is defined by its inability to undergo spontaneous healing over the lifetime of the animal, can be effectively show robust osseous regeneration. This in vivo model demonstrates the basis of translational approaches aimed to regenerate the bone tissue - the cellular component and biological matrix. This method serves as a model for the ultimate clinical application of a progenitor cell towards the repair of a specific tissue defect.

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

Reparatur eines Critical-sized Calvaria Defect Modells mit aus Fettgewebe gewonnenen Stromazellen aus Lipoaspirate Geerntet

Craniofacial skeletal repair and regeneration offers the promise of de novo tissue formation through a cell-based approach utilizing stem cells. ...

Forschung

Medizin

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Mausmodell der Wundheilung

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

Beurteilung von Veränderungen in volatilen Vollnarkose Empfindlichkeit von Mäusen nach lokalen oder systemischen pharmakologischen Intervention

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

Eine einfache Kritische große Femoral Defect Modell an der Maus

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Direkter Maus Trauma / Brennen Modell heterotoper Ossifikationen

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Einrichtung eines segmentalen Femoral Critical-size Defektmodell in Mäusen durch Plattenosteosynthese Stabilisierte

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

Sklerale Vernetzungs Mit Riboflavin und UV-A-Strahlung-für Prävention von Axial Myopie in einem Kaninchenmodell

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

Die Generation der geschlossenen femoralen Frakturen bei Mäusen: ein Modell zur Knochenheilung

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

Fraktur Apparat Design und Protokoll-Optimierung für geschlossen-stabilisierten Frakturen bei Nagern

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

3D Planning and Printing of Patient Specific Implants for Reconstruction of Bony Defects

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical field using the constantly developing 3D planning and printing capabilities. Computer-assisted design (CAD) and computer assisted manufacturing (CAM) are used to describe the 3D planning and manufacturing of the product. The planning and manufacturing of 3D surgical guides and reconstruction implants is performed almost exclusively by engineers. As technology advances and software interfaces become more user-friendly, it raises a question regarding the possibility of transferring the planning and manufacturing to the clinician. The reasons for such a shift are clear: the surgeon has the idea of what he wants to design, and he also knows what is feasible and could be used in the operating room. It allows him to be prepared for any scenario/unexpected results during the operation and allows the surgeon to be creative and express his new ideas using the CAD software. The purpose of this method is to provide clinicians with the ability to create their own surgical guides and reconstruction implants. In this manuscript, a detailed protocol will provide a simple method for segmentation using segmentation software and implant planning using a 3D design software. Following the segmentation and stl file production using segmentation software, the clinician could create a simple patient specific reconstruction plate or a more complex plate with a cradle for bone graft positioning. Surgical guides can be created for accurate resection, hole preparation for proper reconstruction plate positioning or for bone graft harvesting and re-contouring. A case of lower jaw reconstruction following plate fracture and nonunion healing of a trauma sustained injury is detailed.

This protocol describes the use of 3D planning and printing for reconstruction of bony defects. We use segmentation tools to create 3D models followed by 3D design software to create patient specific implants for reconstruction purposes concomitant to ablative surgery or as a second stage.

3D-Planung und Druck von patientenspezifischen Implantaten zur Rekonstruktion von Knochendefekten

We are in the midst of the 3D era in most aspects of life, and especially in medicine. The surgical discipline is one of the major players in the medical ...