Ce travail a été financé par R01-AR053645 de NIAMS.

Toutes les procédures ont été approuvées par le soin des animaux UCSF institutionnel et l&#39;utilisation Comité et se conformer aux directives nationales. 1. Préparation de fixateurs avant la chirurgie <ol><li> Avant de créer une fracture stabilisée, il faut assembler le dispositif sur mesure de conception de fixation externe. Les cadres de fixation sur mesure conçus externes se composait de deux anneaux d&#39;aluminium stabilisés par trois en acier inoxydable # 0/56 tiges filetées, 8 # 2/56 écrous hexagonaux, boulons et 17 correspondants (Partie Petit Inc, Miami Lakes, Floride 3). </li><li> Deux anneaux en aluminium sont nécessaires par fracture. Pour assembler une bague 4 # 2/56 écrous hexagonaux (Partie petite Inc, Miami Lakes, Floride) et des boulons correspondants dans les trous appropriés, ceux-ci devraient rester lâche. Ce sera l&#39;anneau proximal. Pour l&#39;anneau distal assembler 3 écrous et boulons, encore une fois ne pas serrer à fond. </li></ol> 2. Anesthésie, fracture de création, et fixateur demande <ol><li> Souris (âge, sexe, et le corpspoids sont déterminés par l&#39;utilisateur) sont anesthésiés par injection intrapéritonéale de kétamine 50mg/ml, 0,5 mg / ml médétomidine (0,03 ml / souris). </li><li> Appliquer du lubrifiant oeil aux yeux des souris et placez un morceau de ruban adhésif sur leur tête pour protéger leurs yeux lors de la procédure. </li><li> L&#39;animal est placé sur un bloc de chauffage pour le reste de la procédure. </li><li> Transfix les métaphyses proximales et distales du tibia gauche en utilisant quatre broches 0,25 mm insecte stérile (Anticorro: Outil belle science, Foster City, CA) en attachant les chevilles à un outil de Dremel et les percer à travers l&#39;os. </li><li> Repères sont orientés perpendiculairement à l&#39;axe long du tibia, et 90 ° par rapport à l&#39;autre. Au moins 10 mm entre les repères de proximale et distale. </li><li> Placez le premier anneau au-dessus des broches proximales avec les écrous vers l&#39;intérieur, vers les broches. Fixer les broches à l&#39;anneau de maintien du tibia en position centrale au sein de l&#39;anneau en aluminium. </li><li> Placez le second anneau en dessous de la dibroches Stal et sécuriser les broches à l&#39;anneau à l&#39;aide des écrous hexagonaux comme avant. Ajouter le quatrième écrou-boulon combinaison de la partie distale du fixateur. </li><li> Créer la fracture transversale par flexion trois points avec un dispositif approprié. Nous utilisons le système de gravité de Bonnarens et Einhorn 4. Reste la surface postéro-du tibia à travers les blocs, augmenter le poids à une hauteur prédéterminée le long du bras de chute et laisser tomber le poids de telle sorte qu&#39;il en contact avec la face antéro-externe du tibia pour produire la fracture brutale. La hauteur du poids est passé de besoins doit être déterminée pour chaque souche de souris pour assurer une seule fracture du tibia exclusivement. </li><li> Fracture est confirmée par X-ray. </li><li> Après confirmation de la fracture, trois acier inoxydable # 0/56 tiges filetées sont fixés aux anneaux. À partir de l&#39;extrémité distale de la tige et insérer enfiler un écrou sur la bague distale pour bloquer la tige. Ensuite, mettez une autre écrou sur la tige, l&#39;anneau proximal va s&#39;asseoir sur le dessus de ce nut. Veiller à ce que les deux anneaux se tiendra à égale distance et ne sont pas forcer les broches. Fixez l&#39;anneau proximal aux tiges avec un écrou tiers. </li><li> Injections sous-cutanées de la buprénorphine (1,0 mg / kg) pour l&#39;analgésie sont donnés immédiatement après la chirurgie et les animaux sont surveillés pour la douleur et la buprénorphine donnée au besoin. </li><li> L&#39;anesthésie est renversée par l&#39;administration atipamézole, et les animaux sont surveillés jusqu&#39;à ce qu&#39;ils soient adaptés et ambulante. </li></ol> 3. Ostéogenèse par distraction (voir aussi: 12,13) Pour modifier cette procédure pour accueillir la distraction osseuse est simple. Les anneaux sont maintenus en position par des tiges filetées, et en faisant tourner les écrous de maintien des crayons à la place des anneaux peuvent être écartés. <ol><li> Latence: Pour la distraction norme ostéogenèse utilisant ce modèle, les fixateurs sont placés sur le tibia et le laissé seul pendant une période de latence de 5 jours. </li><li> Distraction: osteog distractionenesis peut être effectué en tournant les écrous sur les tiges de stabilisation tour ¼ (0,25 mm) tous les 7 jours jours. </li><li> Elevage: Bony pontage est observé par 10 jours et la consolidation complète est observée par 27 jours après la fin de la distraction. </li></ol> 4. Création d&#39;un défaut critique entreprises Pour créer un défaut critique de taille des fixateurs externes sont appliquées comme décrit précédemment avec les modifications suivantes. <ol><li> Avant de commencer la procédure de la peau est nettoyée avec de l&#39;éthanol 70%. </li><li> Les fixateurs sont appliqués complètement avant de créer le défaut. </li><li> La souris est ensuite placer sous la loupe binoculaire pour visualiser le tibia médial, et une incision (5-7 mm) est réalisé avec une lame # 10 sur un scalpel. </li><li> Le muscle est sectionné et délicatement séparés pour exposer la mi-diaphyse du tibia. </li><li> Un segment de 3 mm de l&#39;os est ensuite retiré de la paire de ciseaux à mi-diaphyse à l&#39;aide. Utilisation de til pointe des ciseaux, l&#39;os est lentement ciselée loin et fragments d&#39;os sont enlevés avec une pince si nécessaire. Des précautions doivent être prises avec les conseils des ciseaux pour éviter de déplacer le tibia avec une grande force mécanique. </li><li> Examiner le site chirurgical pour assurer qu&#39;il n&#39;y a pas de fragments d&#39;os, comme ceux-ci pourraient favoriser la guérison. </li><li> Remplacer le muscle autour des extrémités émoussées du tibia. </li><li> Fermer l&#39;incision sur la peau avec 2-3 points de suture de 6-0 monofilament polyamine. </li><li> L&#39;anesthésie est inversée et les animaux sont suivis pour la douleur et des analgésiques donnée comme décrit précédemment. </li></ol> 5. Les résultats représentatifs Lorsqu&#39;il est correctement appliqué, les fixateurs externes fournissent une plus grande stabilité rigide de la fracture fermée du tibia avec une excellente réduction (fig. 1, 2). Toutefois, dans certains cas, une réduction insuffisante (une lacune évidente et importante entre les extrémités osseuses ou des fractures multiples se produisent (Fig. 3 </strong&gt;), Et ces souris sont exclus des analyses. Fractures stabilisée à l&#39;aide de cette méthode guérir principalement par l&#39;ossification intramembranaire (Fig. 4). En revanche, si la fracture n&#39;est pas stabilisé un cal cartilage grande est formée dans le fossé fracture (Fig. 5), et il est remplacé par de l&#39;os à travers le processus d&#39;ossification membraneuse. <img alt="Figure 1" src="/files/ftp_upload/3552/3552fig1.jpg" /> Figure 1. Radiographies illustrant un dispositif de fixation externe pour stabiliser une fracture du tibia. Radiographie prise après une fracture de montrer quelques segments d&#39;os bien alignées (tête de flèche). <img alt="Figure 2" src="/files/ftp_upload/3552/3552fig2.jpg" /> Figure 2. Image d&#39;une souris après le fixateur a été appliquée. <img alt="Figure 3" src="/files/ftp_upload/3552/3552fig3.jpg" /> Figure 3. Radiographie prise après fracture montre segments osseux mal alignées et fragmentée (tête de flèche). <img alt="Figure 4" src="/files/ftp_upload/3552/3552fig4.jpg" /> Figure 4. Fracture guérit stabilisé par l&#39;intermédiaire d&#39;ossification membraneuse. Coloration trichrome de fracture stabilisée montre un peu d&#39;os nouveau (b) au site de la fracture. Échelle = 500 um. <img alt="Figure 5" src="/files/ftp_upload/3552/3552fig5.jpg" /> Figure 5. Non-stabilisé fracture guérit par l&#39;intermédiaire d&#39;ossification endochondrale. Coloration trichrome de fracture non stabilisée montre cartilage (c) et de l&#39;os (b) au site de fracture. Échelle = 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rockwood and Green's fractures in adults. , 245-271 (2001).</li><li>Garcia, 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+LockingMouseNail-A+New+Implant+for+Standardized+Stable+Osteosynthesis+in+Mice.">The LockingMouseNail-A New Implant for Standardized Stable Osteosynthesis in Mice.</a> J. Surg. Res. , (2009).</li></ol>

Nous n&#39;avons rien à communiquer.

Bones guérir par deux modalités différentes en fonction de la stabilité mécanique (revue dans: 14). Quand on le laisse quelques grandes formes instables modèle du cartilage dans l&#39;espace de fracture qui est remplacé par de l&#39;os pour combler les deux extrémités de l&#39;os cassé. Proximale et distale à la pause, l&#39;os forme directement par l&#39;ossification intramembranaire dans le périoste et endoste. En revanche, dans la guérison des fractures stables se fait exclusivement par l&#39;...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Broches insectes 0.25mm </td><td> Outil belle science </td><td> 26000-25 </td><td> Noirci acier anodisé, 0,25 mm diamètre de la tige, la longueur 4cm </td></tr><tr><td> Écrou acier inoxydable </td><td> Petites pièces </td><td> # 2-56 </td><td> 1/8 &quot;de long, 56 filets au pouce </td></tr><tr><td> Écrou acier inoxydable </td><td> Petites pièces </td><td> # 0-80 </td><td> 1/8 &quot;de long, 80 fils par pouce </td></tr><tr><td> Stainless Screw machine en acier </td><td> Petites pièces </td><td> # 0-80 </td><td> 1/8 &quot;de long, 80 fils par pouce </td></tr><tr><td> Machine en acier inoxydable Couper tige filetée </td><td> Petites pièces </td><td> # 0-80 </td><td> 6 &quot;de longueur, 80 fils par pouce </td></tr><tr><td> En acier inoxydable 18-8 Machine Head Screw </td><td> McMaster-Carr </td><td></td><td> 2-56 Threads, 3/6 &quot;de longueur </td></tr><tr><td> Dispositif de fixation externe </td><td> Atelier d&#39;usinage </td><td> Spécialement conçu </td><td></td></tr></tbody></table>

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

Évaluation des modes de réparation du squelette est essentielle pour le développement de thérapies à être utilisés en clinique pour traiter les fractures. La stabilité mécanique joue un rôle important dans la guérison des lésions osseuses. Dans le pire des cas une instabilité mécanique peut conduire à des êtres humains en retard ou non-union en. Cependant, le mouvement peut aussi stimuler le processus de guérison. Dans les fractures qui ont des formes de mouvement du cartilage pour stabiliser les extrémités osseuses fractures, et ce cartilage est progressivement remplacé par de l&#39;os à travers la récapitulation du processus de développement de l&#39;ossification endochondrale. En revanche, si une fracture de l&#39;os est rigidement stabilisée os forme directement via l&#39;ossification membraneuse. Cliniquement, à la fois l&#39;ossification endochondrale et membraneuse se produisent simultanément. Pour bien répliquer ces enquêteurs processus insérer une broche dans le canal médullaire de l&#39;os fracturé, comme décrit par Bonnarens 4. Cette méthode expérimentale fournit une excellente stabilité latérale tout en permettant rotatiol&#39;instabilité interne de persister. Cependant, notre compréhension des mécanismes qui régulent ces deux processus distincts peuvent également être renforcée par expérimentalement isoler chacun de ces processus. Nous avons développé un protocole de stabilisation qui assure la stabilisation de rotation et latérale. Dans ce modèle, l&#39;ossification intramembranaire est le seul mode de guérison que l&#39;on observe, et les paramètres de guérison peuvent être comparés entre les différentes souches de souris génétiquement modifiées 5-7, après l&#39;application de molécules bioactives 8,9, après avoir changé les paramètres physiologiques de guérison 10, après la modification de la quantité ou le temps de stabilisation 11, après ostéogenèse en distraction 12, après création d&#39;une non-consolidation 13, ou après la création d&#39;un défaut critique taille. Ici, nous illustrons comment appliquer les fixateurs Ilizarov modifiées pour l&#39;étude de la guérison des fractures du tibia et la distraction osseuse chez la souris.

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

Cet article décrit une méthode pour stabiliser les fractures des os longs qui est basé sur l&#39;application de fixateurs externes modifiés Ilizarov<sup&gt; 1-3</sup&gt;. Après l&#39;application des fixateurs et la création de la lésion osseuse, la guérison peut être évaluée, la distraction osseuse peut être effectué, ou non-union ou critiques défaut de taille peut être créé et utilisé pour étudier les interventions thérapeutiques.

Création fractures Rigidement stabilisées pour l&#39;évaluation de l&#39;ossification intramembranaires, ostéogenèse par distraction, ou de guérison des défauts critiques 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

Research

JoVE Journal

Medicine

17.2K vues.  University of California, San Francisco. Cet article décrit une méthode pour stabiliser les fractures des os longs qui est basé sur l'application de fixateurs externes modifiés Ilizarov 1-3. Après l'application des fixateurs et la création de la lésion osseuse, la guérison peut être évaluée, la distraction osseuse peut être effectué, ou non-union ou critiques défaut de taille peut être créé et utilisé pour étudier les interventions thérapeutiques.

Vidéo: 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.

Réparation d&#39;un modèle de critique des défauts de taille crânienne Utilisation adipeuses cellules stromales prélevés sur des Lipoaspirate

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

Recherche

Médecine

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.

Modèle murin de la cicatrisation

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.

Évaluer les changements dans volatils anesthésie générale sensibilité des souris après l&#39;intervention pharmacologique local ou systémique

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.

Une simple critique de taille fémorale défauts modèle chez la souris

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.

Souris Direct Trauma / Graver Modèle d&#39;ossification hétérotopique

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.

Mise en place d&#39;un segmentaire fémoral Critical-size Defect modèle chez la souris Stabilisé par Plate ostéosynthèse

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).

Scléral Cross-linking Utilisation de la riboflavine et Ultraviolet-A rayonnement pour la prévention des Axial Myopie dans un modèle de lapin

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 génération des Fractures fémorales fermées chez la souris : un modèle pour étudier la guérison osseuse

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.

Fracture appareil Design et optimisation de protocoles pour les Fractures fermées-stabilisé chez les rongeurs

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.

Planification et impression 3D d’implants spécifiques aux patients pour la reconstruction des défauts osseux

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 ...