Questo lavoro è stato finanziato dalla R01-AR053645 da NIAMS.

Tutte le procedure sono state approvate dal Institutional Animal Care UCSF e sull&#39;uso Comitato e sono conformi alle linee guida nazionali. 1. Preparazione dei fissatori Prima di Chirurgia <ol><li> Prima di creare fratture stabilizzate, bisogna montare il custom-design del dispositivo di fissazione esterna. Le custom-designed telai esterni di fissaggio costituito da due anelli di alluminio stabilizzata da tre in acciaio inox # 0/56 barre filettate, 8 # 2/56 dadi esagonali e 17 bulloni corrispondenza (Small Part Inc., Miami Lakes, FL 3). </li><li> Due anelli di alluminio sono necessari per frattura. Per assemblare un anello 4 # 2/56 dadi esagonali (piccola parte Inc., Miami Lakes, FL) e bulloni corrispondenti negli appositi fori, questi deve rimanere libera. Questo sarà l&#39;anello prossimale. Per montare l&#39;anello distale 3 dadi e bulloni, ancora una volta non serrare a fondo. </li></ol> 2. Anestesia, Creazione Fracture, e fissatore Application <ol><li> Mice (età, sesso, e il corpopeso sono determinati dall&#39;utente) vengono anestetizzati mediante iniezione intraperitoneale di 50mg/ml chetamina, 0,5 mg / ml medetomidina (0,03 ml / topo). </li><li> Applicare il lubrificante occhio agli occhi dei topi e mettere un pezzo di nastro in tutta la loro testa per proteggere i loro occhi durante la procedura. </li><li> L&#39;animale viene posto su una piastra elettrica per il resto della procedura. </li><li> Trafiggere il metafisi prossimale e distale della tibia sinistra con quattro perni di 0,25 mm di insetti sterili (Anticorro: Strumento Science Fine, Foster City, CA) collegando i pin di uno strumento di Dremel e la perforazione attraverso l&#39;osso. </li><li> Perni sono orientate perpendicolarmente all&#39;asse longitudinale della tibia, e 90 ° fra loro. Almeno 10 mm tra i perni prossimale e distale. </li><li> Posizionare il primo anello sopra i perni prossimali con i dadi rivolti verso l&#39;interno, verso i birilli. Fissare i perni sul ring mantenendo la tibia in posizione centrale all&#39;interno dell&#39;anello di alluminio. </li><li> Posizionare il secondo anello di sotto delperni Stal e fissare i perni per l&#39;anello con i dadi esagonali come prima. Aggiungere il quarto dado-bullone combinazione della distale fissatore. </li><li> Creare la frattura trasversale da tre punti di piegatura con un dispositivo appropriato. Usiamo il sistema di gravità della Bonnarens Einhorn e 4. Appoggiare la superficie postero-laterale della tibia attraverso i blocchi, sollevare il peso ad un&#39;altezza predeterminata lungo il braccio goccia e perdere peso in modo che viene a contatto con l&#39;aspetto antero-laterale della tibia per produrre la frattura smussata. L&#39;altezza del peso viene fatto cadere da deve essere determinato per ogni ceppo di topo per garantire una frattura singola esclusivamente della tibia. </li><li> Fracture è confermata da X-ray. </li><li> Dopo la conferma della frattura, tre acciaio inossidabile # 0/56 aste filettate sono attaccati agli anelli. A partire dall&#39;estremità distale inserire l&#39;asta e infilare un dado sull&#39;anello distale per bloccare l&#39;asta. Poi mettere un altro dado sullo stelo, l&#39;anello prossimale siederà in cima a questo nut. Assicurarsi che i due anelli si terrà a distanze uguali e non sforzare le spine. Fissare l&#39;anello prossimale alle aste con una terza noce. </li><li> Iniezioni sottocutanee di buprenorfina (1,0 mg / kg) per l&#39;analgesia viene dato immediatamente dopo l&#39;intervento chirurgico e gli animali vengono monitorati per dolore e buprenorfina dato quando necessario. </li><li> L&#39;anestesia è invertita con la somministrazione di atipamezolo, e gli animali vengono monitorati fino a quando non sono reattivi e deambulazione. </li></ol> 3. Osteogenesi per distrazione (vedi anche: 12,13) Per modificare questa procedura per accogliere distrazione osteogenesi è semplice. Gli anelli sono tenuti in posizione da barre filettate, e ruotando i dadi di fissaggio delle aste in posizione gli anelli possono essere allontanate. <ol><li> Latenza: Per distrazione osteogenesi standard utilizzando questo modello i fissatori sono posti sul tibia e lasciato solo per un periodo di latenza di 5 giorni. </li><li> Distrazione: osteog Distractionenesis può essere effettuata agendo sui dadi sulle barre stabilizzatrici ¼ di giro (0,25 mm) ogni giorno 7 giorni. </li><li> Maturazione: Bony ponte è osservata da 10 giorni e il consolidamento completo è osservata da 27 giorni dopo il completamento di distrazione. </li></ol> 4. Creazione di un difetto critico Sized Per creare un difetto critico dimensioni dei fissatori esterni vengono applicate come precedentemente descritto con le seguenti modifiche. <ol><li> Prima di iniziare la procedura, la pelle viene pulita con il 70% di etanolo. </li><li> I fissatori sono applicati completamente prima di creare il difetto. </li><li> Il mouse viene quindi posto sotto il microscopio di dissezione per visualizzare la tibia mediale, e una incisione (mm 5-7) è realizzato con una lama # 10 su un bisturi. </li><li> Il muscolo viene delicatamente recisi e separato per esporre la metà diafisi della tibia. </li><li> A 3 mm del segmento osseo viene quindi rimosso dalle forbici utilizzando metà diafisi. Uso tegli punto delle forbici, l&#39;osso è stato tagliato via lentamente e frammenti ossei vengono rimossi con pinze se necessario. Bisogna fare attenzione con le punte delle forbici per evitare di sostituirsi alla tibia con una grande forza meccanica. </li><li> Esaminare il sito chirurgico per garantire che non vi siano frammenti ossei, in quanto potrebbero promuovere la guarigione. </li><li> Sostituire il muscolo intorno alle estremità smussate della tibia. </li><li> Chiudere l&#39;incisione sulla pelle con 2-3 punti di sutura monofilamento 6-0 di poliammine. </li><li> L&#39;anestesia è invertito e gli animali vengono monitorati per dolore e dato analgesici come precedentemente descritto. </li></ol> 5. Risultati rappresentativi Quando correttamente applicato, i fissatori esterni fornire più rigida stabilità della frattura chiusa tibiale con una eccellente riduzione (Figg. 1, 2). Tuttavia, in alcuni casi insufficiente riduzione (una lacuna evidente e grande tra le estremità ossee o fratture multiple verificarsi (Fig. 3 </strong&gt;), E questi topi sono esclusi dalla analisi. Fratture stabilizzato con questo metodo guarire principalmente attraverso ossificazione intramembranosa (Fig. 4). Al contrario, se la frattura non è stabilizzato un callo cartilagine grande è formata nella gap frattura (Fig. 5), e questo è sostituito da osso attraverso il processo di ossificazione intramembranosa. <img alt="Figura 1" src="/files/ftp_upload/3552/3552fig1.jpg" /> Figura 1. Radiografie illustrano un dispositivo esterno di fissaggio utilizzato per stabilizzare frattura tibiale. Radiografia dopo la frattura che mostra alcuni segmenti ossei ben allineati (punta di freccia). <img alt="Figura 2" src="/files/ftp_upload/3552/3552fig2.jpg" /> Figura 2. Immagine di un topo dopo che il fissatore è stato applicato. <img alt="Figura 3" src="/files/ftp_upload/3552/3552fig3.jpg" /> Figura 3. Radiografia presa dopo fracture mostra segmenti ossei non allineate e frammentato (punta di freccia). <img alt="Figura 4" src="/files/ftp_upload/3552/3552fig4.jpg" /> Figura 4. Frattura stabilizzata guarisce via ossificazione intramembranosa. Colorazione tricromica di frattura stabilizzata mostra qualche nuovo osso (b) al sito di frattura. Scala bar = 500 micron. <img alt="Figura 5" src="/files/ftp_upload/3552/3552fig5.jpg" /> Figura 5. Non stabilizzato frattura guarisce via encondrale. Colorazione tricromica di frattura non-stabilizzato mostra cartilagine (c) e osso (b) al sito di frattura. Scala bar = 500 micron.

<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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Non abbiamo nulla da rivelare.

Ossa guarire da due diverse modalità a seconda della stabilità meccanica (recensione in: 14). Se lasciato instabili a grandi forme modello cartilagine nel gap frattura che è sostituito da osso per colmare le due estremità della frattura. Prossimale e distale alla rottura, l&#39;osso si forma direttamente da ossificazione intramembranosa all&#39;interno del periostio e endosteum. Al contrario, in stabile guarigione delle fratture avviene esclusivamente via ossificazione intramembranosa 3. Tuttav...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Insetto pin 0,25 millimetri </td><td> Science ottimo strumento </td><td> 26000-25 </td><td> Oscurati in acciaio anodizzato, asta di 0,25 millimetri di diametro, quattro centimetri di lunghezza </td></tr><tr><td> Dado esagonale in acciaio inossidabile </td><td> Parti piccole </td><td> # 2-56 </td><td> 1/8 &quot;di lunghezza, 56 filetti per pollice </td></tr><tr><td> Dado esagonale in acciaio inossidabile </td><td> Parti piccole </td><td> # 0-80 </td><td> 1/8 &quot;di lunghezza, 80 filetti per pollice </td></tr><tr><td> Vite in acciaio inox della macchina </td><td> Parti piccole </td><td> # 0-80 </td><td> 1/8 &quot;di lunghezza, 80 filetti per pollice </td></tr><tr><td> Macchina in acciaio inox Taglio Rod filettato </td><td> Parti piccole </td><td> # 0-80 </td><td> 6 &quot;lunghezza, 80 filetti per pollice </td></tr><tr><td> 18-8 in acciaio inossidabile Machine Head ScREW </td><td> McMaster-Carr </td><td></td><td> 2-56 Threads &quot;, lunghezza 3/6 </td></tr><tr><td> Fissazione dispositivo esterno </td><td> Officine meccaniche </td><td> Custom-designed </td><td></td></tr></tbody></table>

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

Valutare le modalità di riparazione scheletrica è essenziale per sviluppare terapie per essere usato in clinica per il trattamento di fratture. Stabilità meccanica svolge un ruolo importante nella guarigione delle lesioni ossee. Nel peggiore dei casi instabilità meccanica può portare a ritardi o gli esseri umani non-union in. Tuttavia, il movimento può anche stimolare il processo di guarigione. Nelle fratture che hanno forme di movimento della cartilagine per stabilizzare le estremità della frattura delle ossa, cartilagine e questo viene gradualmente sostituito da tessuto osseo attraverso ricapitolazione del processo di sviluppo di ossificazione endocondrale. Al contrario, se una frattura ossea è rigidamente stabilizzata osso costituisce direttamente tramite ossificazione intramembranosa. Clinicamente, sia encondrale e intramembranosa si verificano simultaneamente. Per replicare efficacemente questo processo investigatori inserire un perno nel canale midollare dell&#39;osso fratturato come descritto da Bonnarens 4. Questo metodo sperimentale fornisce eccellente stabilità laterale consentendo rotazionaleinstabilità NAL a persistere. Tuttavia, la nostra comprensione dei meccanismi che regolano questi due processi distinti può anche essere arricchita da sperimentalmente isolare ognuno di questi processi. Abbiamo sviluppato un protocollo di stabilizzazione che fornisce stabilità rotazionale e laterale. In questo modello, l&#39;ossificazione intramembranosa è l&#39;unica modalità di guarigione che si osserva, ei parametri di guarigione possono essere confrontati tra i diversi ceppi di topi geneticamente modificati 5-7, dopo l&#39;applicazione di molecole bioattive 8,9, dopo alterazione dei parametri fisiologici di guarigione 10, dopo aver modificato la quantità o il tempo di stabilizzazione 11, dopo la distrazione osteogenesi 12, dopo la creazione di un non-union 13, o dopo la creazione di un difetto critico dimensioni. Qui illustriamo come applicare il fissatore Ilizarov modificati per studiare la guarigione della frattura tibiale e la distrazione osteogenesi nei topi.

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

Questo articolo descrive un metodo per stabilizzare fratture delle ossa lunghe che si basa sull&#39;applicazione di modificati Ilizarov fissatori esterni<sup&gt; 1-3</sup&gt;. Dopo l&#39;applicazione dei fissatori e la creazione della lesione ossea, la guarigione può essere valutata, osteogenesi per distrazione può essere eseguita, o non-union o critici difetto dimensioni possono essere creati e utilizzati per studiare interventi terapeutici.

Creare fratture rigidamente stabilizzati per la Valutazione ossificazione intramembranosa, osteogenesi per distrazione, o guarigione di difetti critici Sized

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

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JoVE Journal

Medicine

17.2K Views.  University of California, San Francisco. Questo articolo descrive un metodo per stabilizzare fratture delle ossa lunghe che si basa sull'applicazione di modificati Ilizarov fissatori esterni 1-3. Dopo l'applicazione dei fissatori e la creazione della lesione ossea, la guarigione può essere valutata, osteogenesi per distrazione può essere eseguita, o non-union o critici difetto dimensioni possono essere creati e utilizzati per studiare interventi terapeutici.

Video: 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.

Riparazione di un critico di dimensioni modello Difetto cranica Usando derivate da tessuto adiposo cellule stromali raccolte da Lipoaspirate

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

Ricerca

Medicina

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.

Modello murino di guarigione delle ferite

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.

Valutare Variazioni Volatile generale anestetico sensibilità dei topi dopo l&#39;intervento farmacologico locale o sistemica

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.

Un semplice Critical dimensioni femorale Difetto Modello 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 ...

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.

Diretta mouse Trauma / Burn modello di ossificazione eterotopica

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.

Istituzione di un segmentale femorale critico-size difetto del modello in topi stabilizzati da osteosintesi Piastra

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).

Sclerale reticolazione Utilizzando riboflavina e raggi ultravioletti-A di radiazione per la prevenzione della miopia assiale in un modello di coniglio

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.

La generazione delle fratture chiuse femorale in topi: un modello per studiare la guarigione ossea

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.

Frattura apparato Design e ottimizzazione dei protocolli per le fratture chiuse-stabilizzato in roditori

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.

Pianificazione 3D e stampa di impianti specifici per il paziente per la ricostruzione di difetti di bony

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 ...