Name: direct mouse trauma/burn model of heterotopic ossification
Uploaded: 2015-08-06T14:00:00.000+00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Direct Mouse Trauma/Burn Model of Heterotopic Ossification at JoVE.com

We thank Amanda Fair, the CMI, and Kathy Sweet and the ORL at UM for assistance with &#181;CT imaging and analysis. Funding: BL Funded by 1K08GM109105-01 and Plastic Surgery Foundation National Endowment Award.

Dichiarazione etica: Tutte le procedure sugli animali sono state eseguite in stretta conformità con le buone pratiche animale come definito nelle linee guida fornite nella Guida per l&#39;uso e la cura di animali da laboratorio: Ottava Edizione del Institute for Research Laboratory Animal (ILAR, 2011) e sono stati approvato dalla cura e l&#39;uso degli animali Comitato Istituzionale dell&#39;Università del Michigan (PRO0001553). 1. mouse Procedure chirurgiche NOTA: Usare 8-10 settimane vecchio C57BL / 6 topi. Altre epoche, sfondi e ceppi di topi possono anche essere utilizzati per testare le condizioni diverse o make-up genetico. Per la parte tenotomia del procedimento, mantenere condizioni di sterilità utilizzando la maschera di protezione, cofano capelli, e camice sterile, guanti e strumenti. Siti chirurgici devono essere sterilmente preparati con iodopovidone e drappeggio sterile. Evitare l&#39;ipotermia utilizzando una piastra elettrica veterinaria e il riscaldamento soluzioni rianimazione a 37 ° C prima di amministrazione. <ol><li> Anestetizzare il mouse utilizzando 2,5% isoflurano per via inalatoria. Utilizzare un cono di lasciare il dorso e arti posteriori facilmente accessibile. Regolare la velocità di somministrazione isoflurano per mantenere la frequenza respiratoria adeguata e la colorazione delle mucose e per garantire l&#39;anestesia controllando il tono muscolare, pizzico piedi, e di riflesso corneale. Applicare blando pomata oftalmica agli occhi del mouse per evitare lesioni agli occhi asciutti durante la procedura. Mantenere un&#39;adeguata temperatura corporea usando una piastra elettrica veterinaria o circolatore acqua calda sotto il campo operatorio. </li><li> Somministrare buprenorfina, 0,1 mg / kg, per via sottocutanea immediatamente prima di un intervento chirurgico per l&#39;analgesia. </li><li> Strettamente radere sito chirurgico sulla zampa posteriore sinistra utilizzando tagliaunghie dal tallone al ginocchio. </li><li> Strettamente radere dorso del mouse, iniziando sopra la colonna vertebrale ed estendendo l&#39;area rasata a sinistra della linea mediana dorsale, aprendo una superficie di almeno 2 cm x 3 cm per accogliere il ustioni. </li><li>Preparare il sito chirurgico, compresa la zampa posteriore al ginocchio con 3 alternati scrub povidone-iodio. </li><li> Eseguire una incisione longitudinale lungo la faccia mediale del tendine d&#39;Achille sinistro. Estendere l&#39;incisione in modo che il tendine di Achille può essere facilmente visualizzato; di circa 0,5 cm. </li><li> Eseguire una tenotomia di Achille con tagliente dissezione del tendine a metà con le forbici tessuti taglienti. Inserire una lama delle forbici del tessuto sul piano tessuto sotto il tendine e sezionare lungo il piano finché la lama è al punto medio del tendine. Chiudere le lame a forbice per tagliare il tendine bruscamente. NOTA: Assicurare l&#39;inclusione di tutti i tendini posteriori tra cui gastrocnemio, soleo, e tendini Plantaris. </li><li> Raggiungere emostasi facendo pressione con garza sterile (ci dovrebbe essere minimo sanguinamento) e chiudere l&#39;incisione cutanea con un punto 5-0 Vicryl. </li><li> Eseguire la dorsale parziale spessore di masterizzazione con un blocco di alluminio del peso di 35 g con measuremen approssimativits 2 centimetri x 2 cm x 3 cm riscaldata a 60 ° C in un bagno d&#39;acqua applicata al dorso rasata del mouse per 17 sec. NOTA: ottenere la profondità ustione appropriata posizionando il blocco superiore del mouse anestetizzato, garantendo che l&#39;intera superficie del blocco è in contatto con il mouse, però evitare di applicare qualsiasi ulteriore pressione al blocco, cioè permettono gravità essere l&#39;unica forza tiene il blocco in posizione. Una maniglia di plastica sottile fissato su un lato del blocco è conveniente per la manipolazione, la stabilizzazione, e recupero dal bagno di acqua calda. Questo creerà un organo di totale superficie di circa il 30% a bruciare in un 8-10 settimane di età C57BL / 6 del mouse. Questo ustione contatto è stata scelta rispetto ad altri metodi (fiamma o scottature masterizzazione) a causa della uniformità della profondità dell&#39;ustione sulla ferita e la riproducibilità tra animali. </li><li> Asciugare il sito ustione con una garza e applicare una medicazione Tegaderm. </li><li> Somministrare fluidi riscaldati rianimazione: Ringer lattato soluzione 1 mliniezione intraperitoneale e 0,5 ml iniezione sottocutanea. Questa amministrazione di una volta di rianimazione è adeguata per il recupero da ustioni e tenotomia. NOTA: Non tornare topi anestetizzati per gabbie abitative con altri topi, casa individualmente in gabbie pulite sotto controllo fino a quando i topi sono completamente recuperati. Recupero tipica avviene entro 1-6 ore. Siti di masterizzazione in genere guariscono entro 2-4 settimane e raramente sono complicate da infezione della ferita se tenuti in ambienti abitativi puliti. </li><li> Somministrare buprenorfina 0,1 mg iniezione sottocutanea / kg ogni 12 ore per 3 giorni dopo la procedura. Monitor crescita HO con scansioni seriali μCT a intervalli appropriati. Crescita ossea ectopica è il primo evidente circa 3 settimane dopo l&#39;intervento per μCT. NOTA: La maggior parte dello sviluppo HO è completata entro 9 settimane post-op. Non abbiamo osservato una differenza di volume HO o posizione con ripetute μCT scansioni bi-settimanale per 15 settimane rispetto a un unico end-point μCT scansione a 15 settimane.Al termine degli esperimenti, tutti i topi eutanasia con CO 2 inalazione secondo le linee guida istituzionali e verificare la morte con dislocazione cervicale dopo 10 min. </li></ol> 2. μCT Acquisizione e analisi <ol><li> Fissare il mouse anestetizzato sul piano dello scanner in posizione prona. Nastro arti posteriori saldamente al letto per evitare di respirare artefatti da movimento. Includere l&#39;aria, l&#39;acqua, e idrossiapatite contenente fantasma sotto il mouse per la calibrazione dell&#39;immagine. </li><li> Aprire il software di analisi Bone e definire una regione di interesse (ROI) che comprende entrambi gli arti posteriori dal anca prossimale alla punta della zampa posteriore distale. Ottenere un&#39;immagine utilizzando i seguenti parametri:. 80 kV, 500 μA e 1.300 esposizione msec, 48 micron dimensioni voxel 14,15 </li><li> Calibrare l&#39;immagine di unità Hounsfield (HU) disegnando un ROI in ciascuna delle tre camere fantasma e inserendo la densità media nei campi appropriatiil software. </li><li> Con lo strumento &quot;grabber&quot; nel software, riorientare l&#39;immagine in modo tibia del arto posteriore sinistro è parallelo lungo l&#39;asse Z per consentire la più chiara vista anatomico per la delineazione di strutture ossee corticali ortotopico e HO. </li><li> Cominciando al ginocchio, scorrere distalmente attraverso le fette di immagine fino a quando si incontra HO. Utilizzando lo strumento spline manuale, fare un ROI attorno all&#39;osso ectopica su ogni 5 ° fetta continuando distale attraverso la zampa o fino HO è stato superato. Utilizzare lo strumento estrapolare per estendere e cucire le ROI in un unico ROI che contiene tutte le HO. </li><li> Fare un ROI 3D e scegliere il menu analisi. Calcolare il volume osseo impostando i valori di soglia inferiore e superiore che meglio mostrano la finestra ossea. Utilizzare gli stessi valori soglia fissati per tutte le scansioni. </li></ol>

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B., 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=BMP+type+I+receptor+inhibition+reduces+heterotopic+[corrected]+ossification.">BMP type I receptor inhibition reduces heterotopic [corrected] ossification.</a> Nat Med. 14 (12), 1363-1369 (2008).</li><li>Culbert, A. L., 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=Alk2+regulates+early+chondrogenic+fate+in+fibrodysplasia+ossificans+progressiva+heterotopic+endochondral+ossification.">Alk2 regulates early chondrogenic fate in fibrodysplasia ossificans progressiva heterotopic endochondral ossification.</a> Stem Cells. 32 (5), 1289-1300 (2014).</li><li>Dinther, 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=ALK2+R206H+mutation+linked+to+fibrodysplasia+ossificans+progressiva+confers+constitutive+activity+to+the+BMP+type+I+receptor+and+sensitizes+mesenchymal+cells+to+BMP-induced+osteoblast+differentiation+and+bone+formation.">ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation.</a> J Bone Miner Res. 25 (6), 1208-1215 (1359).</li><li>Peterson, J. R., 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=Burn+injury+enhances+bone+formation+in+heterotopic+ossification+model.">Burn injury enhances bone formation in heterotopic ossification model.</a> Ann Surg. 259 (5), 993-998 (2014).</li><li>Scott, M. A., 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=Brief+review+of+models+of+ectopic+bone+formation.">Brief review of models of ectopic bone formation.</a> Stem Cells Dev. 21 (5), 655-667 (2012).</li><li>Tannous, O., Griffith, C., O'Toole, R. V., Pellegrini, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+after+extremity+blast+amputation+in+a+Sprague-Dawley+rat+animal+model.">Heterotopic ossification after extremity blast amputation in a Sprague-Dawley rat animal model.</a> J Orthop Trauma. 25 (8), 506-510 (2011).</li><li>Tannous, O., 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=Heterotopic+bone+formation+about+the+hip+undergoes+endochondral+ossification:+a+rabbit+model.">Heterotopic bone formation about the hip undergoes endochondral ossification: a rabbit model.</a> Clin Orthop Relat Res. 471 (5), 1584-1592 (2013).</li><li>Peterson, J. R., 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=Treatment+of+heterotopic+ossification+through+remote+ATP+hydrolysis.">Treatment of heterotopic ossification through remote ATP hydrolysis.</a> Sci Transl Med. 6 (255), 255ra132 (2014).</li><li>Peterson, J. R., 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=Early+detection+of+burn+induced+heterotopic+ossification+using+transcutaneous+Raman+spectroscopy.">Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy.</a> Bone. 54 (1), 28-34 (2013).</li><li>Perosky, J. 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A., Forsberg, J. A., Potter, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blast+injuries+and+heterotopic+ossification.">Blast injuries and heterotopic ossification.</a> Bone and Joint Research. 1 (8), 174-179 (2012).</li><li>Hunt, J. L., Arnoldo, B. D., Kowalske, K., Helm, P., Purdue, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+revisited:+a+21-year+surgical+experience.">Heterotopic ossification revisited: a 21-year surgical experience.</a> J Burn Care Res. 27 (4), 535-540 (2006).</li><li>Ring, D., Jupiter, J. 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The authors have nothing to disclose

Heterotopic ossification represents a major functional impairment faced by patients that sustain trauma, burns, and invasive musculoskeletal procedures. The most at-risk population are soldiers in modern conflicts with major blast injuries from mechanisms such as improvised explosive devices (IED).18 Improved body armor and forward positioned medical units allows for improved survival of major extremity injury. After initial stabilization and repair of their extremity injury, these patients are at high risk...

Heterotopic ossification (HO) is the formation of ectopic bone in which osteo-potent cells are aberrantly induced to form endochondral bone outside of the skeleton. While the details of the HO formation pathway are still largely unknown, an accepted paradigm includes three key factors: an inflammatory inciting incident, a permissive niche, and mesenchymal stem cells capable of forming bone.1-3 HO is a common comorbidity complicating over 60% of major burn injuries, 65% of combat-related injuries, and 10% of invasive orthopaedic surgery cases.4,5 However, it is often difficult to predict where HO will form because it can occur at sites of local injury or at distant locations that may be otherwise uninjured. This variability in location makes it difficult to intervene prophylactically to prevent reactive bone formation in a locally targeted manner. There are also congenital forms of HO such as fibrodysplasia ossificans progressiva (FOP) in which patients are prone to the development of robust HO in response to minor trauma or inflammatory insult. Powerful animal models using transgenic mice have reproduced this phenotype and provided insight to the molecular pathways that may also be important in trauma induced HO.6-9 Translational research into the pathogenesis of non-congenital HO has used a wide variety of constructs ranging from injury alone to the implantation of exogenous osteo-inductive materials and/or cells.10-13
In our prior work we have validated a simple and reliable model of HO formation in mice which does not require the administration of any exogenous material.14-17 This model created two key conditions to initiate HO: local trauma and global inflammation. This was achieved through the use of an Achilles tenotomy (local trauma) combined with a distant burn injury (global inflammation). Mice received both treatments concurrently and were found to develop a robust amount of HO that could be analyzed by histologic, radiologic, and molecular means. Interestingly, concurrent burn injury significantly increased the amount of HO that formed and accelerated its developmental time-course.14-16 HO developed at predictable sites around the calcaneus, ankle joint, and tibia/fibula of the limb that received the tenotomy. The reliability of HO development at a known location allowed for focused examination of molecular and histologic features in the early stages of ectopic ossification.14,17 To date, 100% of mice (over 50 animals) with a tenotomy and concurrent burn injury have developed HO. Additionally, longitudinal 2D and 3D imaging and spectroscopic analysis were conducted to examine the growth pattern and biochemical make-up of HO.15,16

<table><tbody><tr><td>C57BL/6 mice</td><td>Jackson Laboratory</td><td>664</td><td>8-10 weeks old</td></tr><tr><td>Isoflurane &amp;ndash; Fluriso</td><td>VET one, Boise, ID</td><td>V1 501017</td><td></td></tr><tr><td>Buprenorphine &amp;ndash; Buprenex</td><td>Reckitt Benckiser Healthcare</td><td>NDC 12496-0757-1</td><td>0.3 mg/ml solution</td></tr><tr><td>Betadine</td><td>Owens and Minor, Mechanicsville, VA</td><td>2047PVP202</td><td></td></tr><tr><td>5-0 Vicryl sutures</td><td>Ethicon, Summerville, NJ</td><td>J493</td><td></td></tr><tr><td>Tegaderm Film, 6 cm x 7 cm</td><td>3M</td><td>1624W</td><td>Cut in half to properly cover burn site</td></tr><tr><td>&amp;micro;CT - GE eXplore Locus SP</td><td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td><td></td><td></td></tr><tr><td>Microview 2.2 Advanced Bone Analysis Application</td><td>GE Healthcare Pre-Clinical Imaging, London, ON, Canada</td><td></td><td></td></tr></tbody></table>

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.

In questo studio, il protocollo è descritto per il modello precedentemente pubblicata murino di trauma / bruciare indotto HO. 14-17 Questo comporta la creazione di una concomitante lesione muscolo-scheletrico localizzata con un tenotomia Achille e insulto infiammatorio globale, con un spessore parziale ustioni. Ciò provoca la formazione di osso affidabile reattivo al sito tenotomia che può essere seguito con l&#39;imaging seriale. Ad oggi, tutti i topi (più di 50) che hanno sostenuto sia Achille tenotomia...

Watch this Scientific Journal Video about Direct Mouse Trauma/Burn Model of Heterotopic Ossification at JoVE.com

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

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 ...

direct-mouse-traumaburn-model-of-heterotopic-ossification

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10.0K Views.  University of Michigan Medical School. 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.

Video: Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.10-15 
Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.16-21
The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.22-30
This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.
The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.
Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. 31
Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. 32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. 37

Pseudofracture, a reproducible murine model of sterile musculoskeletal trauma, allows for evaluation of late term post-traumatic immune responses. This article describes the procedural execution of the model step by step, including the potential for experimental model combinations to permit study of multiple trauma.

Pseudofracture: Una periferica acuta Modello Trauma Tissue

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; ...

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Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Iniezione di cellule di osteosarcoma intratibiale per generare modelli murini di osteosarcoma ortotopico e metastasi polmonari

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Ricerca sul cancro

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.

Un modello di osteogenesi di distrazione del topo

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

Biologia dello sviluppo

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Amputazione e rigenerazione delle cifre del mouse adulto: un modello semplice per indagare sulla formazione del Blastema mammario e sull'ossificazione intramembranosa

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

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 ...

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 ...

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.

Un modello murino di chirurgia ortopedica per studiare la disfunzione cognitiva postoperatoria e rigenerazione tissutale

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

Partial Heterotopic Hindlimb Transplantation Model in Rats

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities after a complex tissue defect. Face and hand transplantations have changed disfigured patients' lives, giving them a new aesthetic and functional social organ. Despite promising outcomes, VCA is still underperformed due to life-long immunosuppression comorbidities and infectious complications. The rat is an ideal animal model for in vivo studies investigating immunological pathways and graft rejection mechanisms. Rats are also widely used in novel composite tissue graft preservation techniques, including perfusion and cryopreservation studies. Models used for VCA in rats must be reproducible, reliable, and efficient with low postoperative morbidity and mortality. Heterotopic limb transplantation procedures fulfill these criteria and are easier to perform than orthotopic limb transplants. Mastering rodent microsurgical models requires solid experience in microsurgery and animal care. Herein is reported a reliable and reproducible model of partial heterotopic osteomyocutaneous flap transplantation in rats, the postoperative outcomes, and the means of prevention of potential complications.

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

Modello di trapianto parziale di arti posteriori eterotopiche nei ratti

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

Immunologia e infezioni

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have ...

Bioingegneria

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Un modello Murine di una ferita da ustione ricostruita con un innesto di pelle allogenica

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...