We thank members of the Mariani lab for critical reading of the manuscript. Our funding sources were: the Baxter Medical Scholar Research Fellowship (to M.K.S.), USC undergraduate fellowships and the Provost, Dean Joan M. Schaeffer, and Rose Hills Fellowships, (to M.K.S.). We also acknowledge a CIRM BRIDGES fellowship through Pasadena City College (to T.T.T). and the James H. Zumberge Research and Innovation Fund, the USC Regenerative Medicine Initiative, and the NIAMS NIH under Award Number R21AR064462 (to F.V.M).

HINWEIS: Alle Verfahren sind in Übereinstimmung mit einem von der Institutional Animal Care und Verwenden Committee (IACUC) an der University of Southern California genehmigten Tier Protokoll. 1. Vorbereitung für die Chirurgie <ol><li> Legen Sie die notwendigen chirurgischen Werkzeuge und Materialien (sterilisiert, Einträge 1 bis 18 in der Materialliste) und vereinbaren Sie einen Binokular und einen Lichtwellenleiter transLicht in einer Haube oder einem Wartungstisch. </li><li> Legen Sie eine geschlechtsreif Maus (20 - 30 g, 1-3 Monate alt) in einer Induktionskammer mit 4% Isofluran Anästhesie zu induzieren. </li><li> Bestätigen Anästhesie mit einer Pfote und / oder Schwanz Prise Test. </li><li> Bewerben Salbe die Augen der Maus die zu Augentrockenheit während der Operation zu vermeiden. </li><li> Bewegen Sie die Maus auf den Mikroskoptisch. Platzieren Sie die Maus in die Bugnase des Wartungsschlauch und seitlich zu positionieren. Geben Sie eine Erwärmung Beutel neben dem Tier zur Aufrechterhaltung der Körpertemperatur. NICHTE:. Position der Maus hängt von persönlichen Vorlieben und / oder Händigkeit des Chirurgen, der rechten oder linken Seite ist in Ordnung. </li><li> Passen Isofluran zu 2-3% für die Wartung, und beobachten Sie die Atemfrequenz. </li><li> Injizieren Buprenorphin (0,05 mg / kg) subkutan (Seitenflanke des Beins) zum postoperativen Schmerz mit einer 25 G-Nadel. Das Bein kann zucken, welche eine normale Reaktion ist. </li></ol> 2. Öffnungs Incision <ol><li> Ermitteln Sie die gewünschte Rippe durch Palpation, und löschen Sie den Bereich der Haare mit einem Rasierer. HINWEIS: Rippen 8-10 werden empfohlen, da Rippen 1-7 sind näher an der Lunge und 11-13 sind deutlich kürzer. Zählen der Rippen sollte mit den meisten rostral Rippe als Nummer 1 beginnen. </li><li> Bereiten Sie die Gegend mit wechsel PVP-Jod und Isopropanol (70%) Abtupfen (3x wird oft von Tiernutzung Protokolle empfohlen). </li><li> Schneiden Sie ein 2 cm Schnitt durch die Haut, direkt über und parallel mit mittelgroßen Mikroscheren gewünschten Rippe. IncisE durch die darunter liegende Muskulatur und Fettschichten. </li><li> Legen Sie alle drei Schichten (Haut, Muskeln, Fett) in eine Rückziehvorrichtung, um das Operationsfeld freizulegen, bei gleichzeitiger Minimierung der Größe des Einschnitts. </li></ol> 3. Ausschneiden des Rib <ol><li> Durch die Zwischenrippenmuskeln, die über dem gewünschten Abschnitt der Rippe mit einer 5,0 mm Skalpell. Position diesen Schnitt um 5 mm proximal der chondrocostal gemeinsame da hier die Rippe ist nicht so stark gekrümmten. Trennen Sie vorsichtig die Muskeln von den Knochen mit feiner Spitze Pinzette. </li><li> Um eine Resektion zu schaffen unter Beibehaltung der Knochenhaut in der Tier, schneiden durch die Knochenhaut, über die Länge der Rippe mit einer 5,0 mm Skalpell. Trennen Sie vorsichtig das Periost von der darunter liegenden Knochen seitlich feine Spitze Pinzette. Gehen Sie mit Vorsicht, da die Knochenhaut ist sehr empfindlich und hat eine gelartige Konsistenz. </li><li> Weiter stellen Sie einen Querschnitt durch den Knochen an einem Ende mit feinen Mikro Schere. Bei Bedarf messen die Resektion mitein Fadenkreuz im Mikroskop oder einem Lineal Typ Wertangabe. Dann heben Sie die Knochen von der Knochenhaut und schneiden Sie das andere Ende. HINWEIS: Gehen Sie mit Vorsicht. Dies ist der heikelste Schritt, wie oben ziehen, ohne Auslösen der Rippe unter die Pleura Membran reißen und zu einem Pneumothorax. Wenn der pleuralen Membran zerrissen wird dies ohne weiteres ersichtlich sein, wie das Innere der Brusthöhle sichtbar werden. </li><li> Wenn die Blutung als Folge des Schneidens des Knochens auftritt, wenden Sie Druck an der Schnittende mit einem Wattestäbchen für 4-5 Sekunden, um die Blutung zu stoppen. HINWEIS: In den seltenen Fällen, dass Blutung anhält, kann es notwendig sein, zu stoppen und das Tier einschläfern als signifikante Blutverlust wird die Erholung gefährden (ohne Flüssigkeitsersatz ist die maximale Blutvolumen, die sicher verloren gehen können 10% des gesamten Blutvolumens oder 7.7 -. 8 ul / g für eine 25 g Maus entspricht dies rund 180 bis 200 &amp; mgr; 9). </li><li> Sofort legen Sie die entfernt Rippe in4% PFA für eine spätere Analyse. HINWEIS: Wenn die Resektion von sowohl dem Knochen und der Knochenhaut ist durchgeführt werden soll, lassen Sie den Knochenhaut Schnitt und Trennung (Schritt 3.2). Da die Knochenhaut ist sehr eng an die Pleura Membran angebracht gehen vorsichtig, Hänseleien der Rippe von der Pleura-Membran mit einer Pinzette, um Risse zu vermeiden. </li></ol> 4. Schließen der Inzision <ol><li> Nähen Sie die Zwischenrippenmuskeln über die Spitze des verbliebenen periostalen Hülle mit 9-0 Nahtmaterial (2 Nähte sind in der Regel ausreichend). Zeigen die Fäden direkt über die Schnittenden der Rippe, die als Indikatoren für die Chirurgie Ort handeln. </li><li> Entfernen Sie die Rückziehvorrichtung. Naht der darüberliegenden Muskeln und Fett mit 9-0 Nahtmaterial (3-4 Nähte sind in der Regel ausreichend). In Mäusen, die mit besonders dicken Schichten von Muskulatur und / oder Fett, Naht jede Ebene (beispielsweise 1-Schicht von Nähten für die Muskelschicht und 1 Schicht von Nähten für die Fettschicht). </li><li> Schließen Sie dieHaut mit 7-0 Nahtmaterial (4-5 Nähte sind in der Regel ausreichend). </li><li> Sichern Sie den Schnitt mit Nahtkleber, Kneifen die Ränder zusammen mit großen Zangen. </li><li> Langsam zu entwöhnen Sie die Maus aus Isofluran durch zunächst für einige Minuten Einstellung auf 1% und schalten Sie. </li><li> Platzieren Sie die Maus unter einer Wärmelampe und verlassen, bis das Bewusstsein wiedererlangt, 5 - 10 min. Nach Bewusstsein wiedererlangt und während der Heilungsphase sollte die Maus bewegen und normal ambulate und zeigen keine Anzeichen von Leiden. </li></ol> 5. Wiederaufnahme und Analyse <ol><li> Geben postoperativen Schmerztherapie einschließlich der Anwendung von Buprenorphin in einer mündlichen Gelatineform bei 0,5 mg / kg alle 12 Stunden für 48 Stunden. Die orale Verabreichung erfordert keine Zurückhalten der Tiere, die Schmerzen für den Brustbereich verursachen könnten. </li><li> Pflegen Sie die Maus in einen Käfig mit freiem Zugang zu Futter und Wasser während der Heilungsphase. Nachdem der Schnitt trocken ist, können weibliche Tiere zusammengebracht werden, während Männers müssen getrennt bleiben, um Kämpfe zu vermeiden. </li><li> Nach der Einheilzeit, folgen Sie dem zugelassenen Verfahren für die Euthanasie. Entfernen Sie den Brustkorb zur Fixierung und Analyse. </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 21 (4), 685-690 (2003).</li><li>Matthys, R., Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+fixator+for+use+in+the+mouse.">Internal fixator for use in the mouse.</a> Injury. 40, S103-S109 (2009).</li><li>Cooper, G. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+somitic+cells+to+the+avian+ribs.">Contribution of somitic cells to the avian ribs.</a> Developmental biology. 256 (1), 114-126 (2003).</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 (17), 4123-4133 (2003).</li><li>Lu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+basis+for+age-related+changes+in+fracture+repair.">Cellular basis for age-related changes in fracture repair.</a> Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 23 (6), 1300-1307 (2005).</li><li>Zilberman, Y., Gafni, Y., Pelled, G., Gazit, Z., Gazit, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+imaging+in+bone.">Bioluminescent imaging in bone.</a> Methods in molecular biology. 455, 261-272 (2008).</li><li>Pelled, G., Gazit, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+using+osteocalcin-luciferase.">Imaging using osteocalcin-luciferase.</a> Journal of musculoskeletal. 4 (4), 362-363 (2004).</li><li>Elefteriou, F., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+for+bone+studies--strengths+and+limitations.">Genetic mouse models for bone studies--strengths and limitations.</a> Bone. 49 (6), 1242-1254 (2011).</li><li>Srour, M. 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The authors have nothing to disclose

Wenn erst zu lernen, dieses Protokoll, zu bestimmen, wo die anfänglichen Schnitt zu finden kann eine Herausforderung sein. , Praxis auf eingeschläfert Mäusen hilft jedoch dem Chirurgen erfahren, wo die anfänglichen Schnitt legen und setzen die gewünschte Rippe reseziert werden. Die Arbeit an Kadavern verbessert auch die feinmotorischen Fähigkeiten erforderlich, um den Rippenabschnitt mit oder ohne Knochenhaut zu entfernen. Zudem könnte jemand neu in diesem Verfahren finden die Manipulation der feinen Werkzeuge un...

Schwächenden Skelettverletzungen, chronische Arthrose, und die schwere Probleme mit der Wiederherstellungschirurgie verbunden Auswirkungen der wirtschaftlichen Produktivität, Familie Wohlbefinden und Lebensqualität. Während kleine Pausen und Läsionen können ziemlich gut heilen, sind die Menschen nicht in der Lage zu reparieren große Defekte und muss daher auf rekonstruktive Eingriffe angewiesen, um Struktur und Funktion wiederherzustellen. Die Rekonstruktion kann es sich um allogene Transplantate oder heterogeneic, morcellized Knochen implantiert Gerüste oder Distraktionsosteogenese. Leider gibt es nicht nur hartnäckig Morbidität Faktoren mit diesen Behandlungen aber die ursprüngliche Festigkeit des reparierten Knochens selten erreicht verbunden. So werden neue klinische Ansätze erforderlich. One-way, um innovative Methoden, um Segmentdefekte zu behandeln entwickeln zu Situationen, in denen groß angelegte Reparatur kommt in der Natur zu studieren. Amphibien können bekanntlich Skelettelemente zu regenerieren, während Säugetiere sind als begrenzt in thist die Fähigkeit. Da jedoch der Beginn des 20. Jahrhunderts, einige wenige Berichte über die Regeneration der menschlichen Rippe wurden veröffentlicht hindeutet, dass Menschen nicht so beschränkt 1-4 werden. Derzeit wird dieses Phänomen am besten plastischen Chirurgen, die Rippenmaterial für Kiefer-, Gesichts- und Gehör Rekonstruktion verwenden bekannt, aber es ist nicht mehr weit geschätzt 5. Um dieses Reparatur genauer zu untersuchen, haben wir ein chirurgisches Modell unter Verwendung der Maus entwickelt. Mit diesem Protokoll können die Forscher die angeborenen Faktoren zu identifizieren und diese Informationen benutzen, um Skelettheilung an anderen Standorten zu erleichtern. Es gibt viele Vorteile, um mit den Rippen als Modell für die Untersuchung der Skelettreparatur. Zunächst werden die umgebenden Rippen eine natürliche Fixateur (verglichen mit der Resektion des Femurs 6,7). Dies verringert das Morbiditätsrisiko der internen und externen Fixatoren und vereinfacht die chirurgische Prozedur. Zweitens sind die dünnen Muskelschichten der Brust wawerde sorgen für einfachen Zugang und gute Sicht, die der Test vergleichbar mit dem Komfort der Schädel Resektionen 8 zu machen. Drittens, im Gegensatz zu der, die von Calvarien intramembranous Ossifikation Rippen Form durch endochondrale Ossifikation bilden und über Verlängerung an Wachstumszonen an jedem Ende eines zentralen Diaphyse angeordnet wachsen in der Länge. Daher kann die Reparatur der Rippen mehr vergleichbar Reparatur der langen Röhrenknochen der Gliedmaßenskelett sein. Darüber hinaus haben wir festgestellt, dass im Vergleich zu dem Femur, die Knochenhaut der Rippe dicker und kann leichter gehandhabt werden. So können Forscher, die zur Knochenreparatur zum Zweck der Untersuchung der Knochenhaut oder Testzelltherapien, pharmakologische Wirkstoffe und / oder Gewebegerüste testen möchten finden diese Operationsmodell sinnvoll. Zusammenfassend stellt diese Rippenresektion Modell ein Rahmen ist, um natürliche großen Knochenreparatur bei Säugetieren zu studieren, da kein solches Modell im allgemeinen Gebrauch derzeit existiert.

<table><tbody><tr><td>Medium sized micro-dissection scissors (Vannas-T&amp;uuml;bingen Spring Scissors 5 mm)</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)</td><td>Fine Science Tools</td><td>15000-04</td><td>curved tip is beneficial</td></tr><tr><td>Micro-scalpel 5.0 mm</td><td>Fine Science Tools</td><td>10315-12</td><td>other fine scalpels can be substituted</td></tr><tr><td>Dumont 55 forceps</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Retractor&amp;nbsp;</td><td>Fine Science Tools</td><td>17004-05</td><td>adjustability is convenient</td></tr><tr><td>Micro-needle holders</td><td>Fine Science Tools</td><td>12060-01</td><td></td></tr><tr><td>9.0 nylon sutures (Ethilon), taper point best</td><td>Ethicon</td><td>2819G or similar</td><td>taper point best but reverse cutting is also good</td></tr><tr><td>7.0 prolene sutures (Prolene)</td><td>Ethicon</td><td>8700H or similar</td><td>6-0 can be used too, needle point can vary</td></tr><tr><td>Large forceps (Adson Forceps)</td><td>Fine Science Tools</td><td>11006-12</td><td>other brands are fine</td></tr><tr><td>Lubricant Eye Ointment (Akwa Tears)</td><td>Akorn</td><td>17478-062-35</td><td></td></tr><tr><td>Suture glue (GLUture Topical Tissue Adhesive)</td><td>Abbot</td><td>32046-01</td><td>has excellent working time</td></tr><tr><td>Shaver</td><td>Wahl</td><td>9918-6171 or similar</td><td></td></tr><tr><td>Clamp lamp</td><td>Zoo Med</td><td>LF-5</td><td></td></tr><tr><td>Infrared Bulb, 75W</td><td>Zoo Med</td><td>RS-75</td><td></td></tr><tr><td>RC&lt;sup&gt;2&lt;/sup&gt; Rodent Anesthesia System</td><td>VetEquip&amp;nbsp;</td><td>922100</td><td></td></tr><tr><td>IsoFlo (Isoflurane)</td><td>Abbot</td><td>05260-05</td><td></td></tr><tr><td>Buprenorphine (Buprenex)</td><td>Reckitt Benckiser</td><td>12496-0757-1</td><td></td></tr><tr><td>Betadine</td><td>Purdue Frederick</td><td>67618015017</td><td></td></tr><tr><td>Flavored Gelatin, raspberry</td><td>Jell-O</td><td>B000E1FYL0</td><td>made up firm, to the consistency of &#039;jigglers&#039;</td></tr></tbody></table>

a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair

Dieses Protokoll stellt die Forscher ein neues Modell für große Knochenreparatur unter Verwendung der Maus Rippe. Das Verfahren beschreibt die folgenden: Vorbereitung des Tieres für die Chirurgie, Öffnen des Brustkörperwand, indem die gewünschte Rippe aus den umliegenden Zwischenrippenmuskeln, Ausschneiden des gewünschten Abschnitt der Rippe, ohne einen Pneumothorax, und Schließen der Schnitte. Im Vergleich zu den Knochen des Gliedmaßenskelett, die Rippen sind sehr zugänglich. Darüber hinaus ist kein interner oder externer Fixateur notwendig, da die benachbarten Rippen eine natürliche Fixierung. Die Operation nutzt kommerziell verfügbare Angebot, ist einfach zu lernen, und durch das Tier gut vertragen. Das Verfahren kann mit oder ohne Entfernung des umgebenden Knochenhaut erfolgen, und somit ist der Beitrag der Knochenhaut zu reparieren beurteilt werden kann. Die Ergebnisse zeigen, dass, wenn die Knochenhaut gehalten wird, in 1 tritt robust Reparatur - 2 Monate. Wir gehen davon aus, dass die Verwendung dieses Protokolls wirdFörderung von Forschung in die Rippe Reparatur und dass die Ergebnisse wird die Entwicklung von neuen Wegen, um die Knochenreparatur in anderen Orten rund um den Körper zu stimulieren, zu erleichtern.

Tiere in der Regel schnell wieder von diesem Verfahren, der Schnitt heilt gut, und die Tiere zeigen normales Verhalten. Die Prozedur verwendet handelsübliche Versorgung und nach dem Training, kann in weniger als 1 Stunde durchgeführt werden. Bevor Sie dieses Verfahren, um experimentelle Daten zu generieren, ist es wichtig, die Resektion am Tag 0 Zeitpunkt zu analysieren, um festzustellen, ob große Knochenfragmente könnte innerhalb der Resektion Zone bleiben. Ein Weg, um die richtige Vorgehensweise zu beurteilen ist,...

Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Ein chirurgischer Eingriff zur Resektion der Maus Rib: Ein Modell für Großlangknochenreparatur

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

a-surgical-procedure-for-resecting-mouse-rib-model-for-large-scale

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Medicine

13.1K Aufrufe.  Keck School of Medicine, University of Southern California. The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Serie: A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

A Mouse Distraction Osteogenesis Model

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the following three phases: the latency phase after osteotomy and placement of the external distractor; the distraction phase, wherein the separated bone ends are gradually and continuously distracted; and the consolidation phase. This custom-made distractor used for DO is comprised of two incomplete acrylic resin rings and an expansion screw. The process was initiated by making a mold with silicone impression material and then creating the custom-made distractor. Dental resin was poured into the formwork made of silicone impression material, and it was allowed to polymerize to create the incomplete resin rings required for the custom-made distractor. These rings were fixed with an expansion screw using transparent resin. The custom-made distractor created via this approach was attached to the tibia of mice. The tibia was fixed to the device using one pair of 25 G needles proximally, one pair of 27 G needles distally, and acrylic resin. After a latency period of 5 days, distraction was initiated at a rate of 0.2 mm/12 h. The lengthening was continued for 8 days, resulting in a total gap of 3.2 mm. The mice were sacrificed 4 weeks after distraction. Bone formation in the distraction gap was confirmed using both radiography and histology.

We present a mouse tibial distraction osteogenesis model developed using a custom-made distractor. The use of a mouse as an analysis target is advantageous for advancing research.

Ein Mausmodell Ablenkung Osteogenesis

Distraction osteogenesis (DO) is a surgical procedure that involves skeletal tissue regeneration without cell transplantation. A DO model consists of the ...

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Entwicklungsbiologie

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

Maus Pneumonektomie Modell der Ausgleichs Lung Wachstum

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Medizin

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 ...

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 ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

Ein Maus-Modell der orthopädischen Chirurgie, Postoperative kognitive Dysfunktion und Geweberegeneration zu studieren

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

Eine Minimal-Invasive Modell Endochondral Frakturheilung bei Mäusen unter standardisierten Bedingungen biomechanische Analyse

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

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 ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

Querbruch des Mausfemurs mit stabilisierendem Stift

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

Biologie

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

Etablierung eines diaphysären Femurfrakturmodells bei Mäusen

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...

3D-Visualisierung des Knochenumbaus unter Zugbelastung bei Nahtexpansionsmäusen

A 3-D Visualization Technique for Bone Remodeling in a Suture Expansion Mouse Model

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and facial bone growth, housing mesenchymal stem cells and osteoprogenitors. As most craniofacial bones develop through intramembranous ossification, the sutures' marginal regions act as initiation points. Due to this significance, these sutures have become intriguing targets in orthopedic therapies like spring-assisted cranial vault expansion, rapid maxillary expansion, and maxillary protraction. Under orthopedic tracing force, suture stem cells are rapidly activated, becoming a dynamic source for bone remodeling during expansion. Despite their importance, the physiological changes during bone remodeling periods remain poorly understood. Traditional sectioning methods, primarily in the sagittal direction, do not capture the comprehensive changes occurring throughout the entire suture. This study established a standard mouse model for sagittal suture expansion. To fully visualize bone remodeling changes post-suture expansion, the PEGASOS tissue clearing method was combined with whole-mount EdU staining and calcium chelating double labeling. This allowed the visualization of highly proliferating cells and new bone formation across the entire calvarial bones following expansion. This protocol offers a standardized suture expansion mouse model and a 3-D visualization method, shedding light on the mechanobiological changes in sutures and bone remodeling under tensile force loading.

Autor im Rampenlicht: PEGASOS Tissue Clearing-Technik zur Visualisierung des Knochenumbaus 

This protocol presents a standardized suture expansion mouse model and a 3-D visualization method to study the mechanobiological changes of the suture and bone remodeling under tensile force loading.

Eine 3D-Visualisierungstechnik für den Knochenumbau in einem Nahtexpansions-Mausmodell

Craniofacial sutures play a crucial role beyond being fibrous joints connecting craniofacial bones; they also serve as the primary niche for calvarial and ...