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.

伦理声明:所有动物的程序进行，严格按照定义的指南实验动物的使用和注意事项中提供的准则良好做法的动物:第八版从学院实验动物研究（ILAR，2011）和人经密歇根大学的机构动物护理和使用委员会（PRO0001553）。 1.鼠标外科手术注意:使用8-10周龄C57BL / 6小鼠。其他年龄，背景，和小鼠的菌株也可用于测试不同的条件或遗传组成。对于该过程的切断术部，保持用面罩，吹风盖和无菌长袍，手套，和仪器无菌条件。手术部位应该用碘伏消毒，并进行立体裁剪无菌准备。通过使用兽医加热垫和升温复苏溶液至37℃，避免低温之前administra化。 <ol><li>麻醉，用2.5％的异氟烷​​吸入鼠标。用鼻锥离开背部和后肢肢体方便。调整异氟烷给药速度保持粘膜适当呼吸速率和着色，并确保麻醉通过检查肌肉张力，脚趾捏和角膜反射。应用平淡眼药膏鼠标的眼睛，以防止在手术过程中干眼的伤害。采用外科领域的下方兽医加热垫或温水循环保持足够的体温。 </li><li>辖丁丙诺啡，0.1毫克/千克，皮下之前立即手术镇痛。 </li><li>密切刮胡子使用指甲刀从脚跟到膝盖左侧后爪的手术部位。 </li><li>密切剃鼠标的背部，开始在脊柱和剃区域延伸到背中线的左边，结算的面积至少为2厘米×3cm至容纳烫伤。 </li><li>准备手术部位，包括后爪来使用3-交替碘伏磨砂膝盖。 </li><li>沿左跟腱的内侧面进行纵形切口。延长切口所以跟腱可以容易地可视化;约为0.5公分。 </li><li>在用锋利的剪刀组织的中点执行跟腱腱切断术与肌腱锐性剥离。插入所述组织剪刀的一个叶片在所述组织平面肌腱下方并沿平面解剖直到刀片是在肌腱中点。关闭剪刀片大幅割断肌腱。 注:确保所有列入筋后腓肠肌包括，比目鱼肌，跖肌腱。 </li><li>达到止血施加压力，用无菌纱布（应该有最小的出血）和5-0薇乔缝合关闭皮肤切口。 </li><li>与铝块重达35克与近似measuremen进行背Ⅱ度烧伤TS2厘米×2厘米×3厘米施加到小鼠的剃毛背部为17秒的水浴加热到60℃。 注:实现适当的烧伤深度由搁在块上的麻醉小鼠的顶部，确保该块的整个表面区域是在用鼠标接触，但是避免施加任何额外的压力来将挡， 即，允许重力将唯一力捧到位块。附连到块的一侧的薄塑料手柄便于操纵，稳定化，并从热水浴检索。这将创建在8-10周龄C57BL / 6小鼠的约30％体表面积烧​​伤。这种接触烧伤被选择了其他的方法（火焰或烫伤烧伤）因烧伤深度的均匀性伤口和动物之间的重现性。 </li><li>干烧伤部位用纱布和应用TEGADERM敷料。 </li><li>辖回暖复苏液:乳酸林格氏溶液1ml腹腔注射和0.5ml皮下注射。这一次复苏的管理是足够的距离烧伤和切断术恢复。 注意:不要单独在干净的笼子下监视返回麻醉小鼠住房笼与其他老鼠，房子，直到小鼠完全恢复。典型的恢复发生在1-6小时。烧伤部位一般在2-4周内自愈，很少通过伤口感染复杂，如果存放在干净的环境中的住房。 </li><li>辖丁丙诺啡0.1毫克/千克皮下注射，每12小时3天以下的步骤。何监督增长，串行μCT扫描在适当的间隔。异位骨的生长是第一明显约3周后运通过μCT。 注:大多数HO发展完成了9周后运。我们没有观察到何体积或反复μCT位置的差扫瞄每两周15周相比单终点μCT在15周扫描。在实验结束时，安乐死所有小鼠用CO 2吸入根据机构指南和10分钟后确认死亡颈椎脱位。 </li></ol> 2.μCT采集与分析<ol><li>确保麻醉鼠标放在扫描仪床上俯卧位。大盘后肢牢固地固定在床上，以防止呼吸运动伪影。包括空气，水，和羟基磷灰石含有小鼠用于图像校正的下方幻象。 </li><li>打开骨分析软件并定义感兴趣区域（ROI），它包括从髋关节都后肢近侧到后爪向远侧的末端的区域。使用以下参数获得图像:80千伏，500微安和1300毫秒曝光，48微米的体素尺寸14,15 </li><li>通过绘制的投资回报率在每三个幻影室，并输入到相应的字段的平均密度校准图像菲尔德单位（HU）该软件。 </li><li>使用在软件的"抓取器"工具，重新定向的图像，从而使左后肢的胫骨是沿Z轴平行，以允许原位皮质骨结构和HO划定清亮的解剖图。 </li><li>在膝盖开始，通过图像切片远端滚动，直到何遇到。使用手动样条曲线工具，使周围的异位骨的投资回报，每5 个片通过爪或直到何已持续超越的远端。使用外推工具来扩展和缝合的投资回报率连成一片的投资回报率是包含了所有的议员。 </li><li>做一个3D的投资回报率，然后选择菜单分析。通过设定下和上阈值的最佳显示骨窗计算骨体积。使用相同的固定阈值的所有扫描。 </li></ol>

<ol><li>Leblanc, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BMP-9-induced+muscle+heterotopic+ossification+requires+changes+to+the+skeletal+muscle+microenvironment%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment.</a> J Bone Miner Res. 26 (6), 1166-1177 (2011).</li>
<li>Shore, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoinductive+signals+and+heterotopic+ossification%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">Osteoinductive signals and heterotopic ossification.</a> J Bone Miner Res. 26 (6), 1163-1165 (2011).</li>
<li>Wosczyna, M. N., Biswas, A. A., Cogswell, C. A., Goldhamer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multipotent+progenitors+resident+in+the+skeletal+muscle+interstitium+exhibit+robust+BMP-dependent+osteogenic+activity+and+mediate+heterotopic+ossification%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">Multipotent progenitors resident in the skeletal muscle interstitium exhibit robust BMP-dependent osteogenic activity and mediate heterotopic ossification.</a> J Bone Miner Res. 27 (5), 1004-1017 (2012).</li>
<li>Potter, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterotopic+ossification+following+combat-related+trauma%5BTitle%5D)+AND+%22J+Bone+Joint+Surg+Am%22%5BJournal%5D)">Heterotopic ossification following combat-related trauma.</a> J Bone Joint Surg Am. 92, Suppl 2. 74-89 (2010).</li>
<li>Van den Bossche, L., Vanderstraeten, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterotopic+ossification%3A+a+review%5BTitle%5D)+AND+%22J+Rehabil+Med%22%5BJournal%5D)">Heterotopic ossification: a review.</a> J Rehabil Med. 37 (3), 129-136 (2005).</li>
<li>Chakkalakal, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+Acvr1+R206H+knock-in+mouse+has+fibrodysplasia+ossificans+progressiva%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva.</a> J Bone Miner Res. 27 (8), 1746-1756 (2012).</li>
<li>Yu, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BMP+type+I+receptor+inhibition+reduces+heterotopic+%5Bcorrected%5D+ossification%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">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/pubmed?term=((Alk2+regulates+early+chondrogenic+fate+in+fibrodysplasia+ossificans+progressiva+heterotopic+endochondral+ossification%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22J+Bone+Miner+Res%22%5BJournal%5D)">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/pubmed?term=((Burn+injury+enhances+bone+formation+in+heterotopic+ossification+model%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">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/pubmed?term=((Brief+review+of+models+of+ectopic+bone+formation%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">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/pubmed?term=((Heterotopic+ossification+after+extremity+blast+amputation+in+a+Sprague-Dawley+rat+animal+model%5BTitle%5D)+AND+%22J+Orthop+Trauma%22%5BJournal%5D)">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/pubmed?term=((Heterotopic+bone+formation+about+the+hip+undergoes+endochondral+ossification%3A+a+rabbit+model%5BTitle%5D)+AND+%22Clin+Orthop+Relat+Res%22%5BJournal%5D)">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/pubmed?term=((Treatment+of+heterotopic+ossification+through+remote+ATP+hydrolysis%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">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/pubmed?term=((Early+detection+of+burn+induced+heterotopic+ossification+using+transcutaneous+Raman+spectroscopy%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Early detection of burn induced heterotopic ossification using transcutaneous Raman spectroscopy.</a> Bone. 54 (1), 28-34 (2013).</li>
<li>Perosky, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+detection+of+heterotopic+ossification+using+near-infrared+optical+imaging+reveals+dynamic+turnover+and+progression+of+mineralization+following+Achilles+tenotomy+and+burn+injury%5BTitle%5D)+AND+%22J+Orthop+Res%22%5BJournal%5D)">Early detection of heterotopic ossification using near-infrared optical imaging reveals dynamic turnover and progression of mineralization following Achilles tenotomy and burn injury.</a> J Orthop Res. 32 (11), 1416-1423 (2014).</li>
<li>Peterson, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Aging+on+Osteogenic+Response+and+Heterotopic+Ossification+Following+Burn+Injury+in+Mice%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">Effects of Aging on Osteogenic Response and Heterotopic Ossification Following Burn Injury in Mice.</a> Stem Cells Dev. , (2014).</li>
<li>Alfieri, K. A., Forsberg, J. A., Potter, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blast+injuries+and+heterotopic+ossification%5BTitle%5D)+AND+%22Bone+and+Joint+Research%22%5BJournal%5D)">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/pubmed?term=((Heterotopic+ossification+revisited%3A+a+21-year+surgical+experience%5BTitle%5D)+AND+%22J+Burn+Care+Res%22%5BJournal%5D)">Heterotopic ossification revisited: a 21-year surgical experience.</a> J Burn Care Res. 27 (4), 535-540 (2006).</li>
<li>Ring, D., Jupiter, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Operative+release+of+ankylosis+of+the+elbow+due+to+heterotopic+ossification.+Surgical+technique%5BTitle%5D)+AND+%22J+Bone+Joint+Surg+Am%22%5BJournal%5D)">Operative release of ankylosis of the elbow due to heterotopic ossification. Surgical technique.</a> J Bone Joint Surg Am. 86-A, Suppl 1. 2-10 (2004).</li>
<li>Crane, N. J., Polfer, E., Elster, E. A., Potter, B. K., Forsberg, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Raman+spectroscopic+analysis+of+combat-related+heterotopic+ossification%5BTitle%5D)+AND+%22Bone%22%5BJournal%5D)">Raman spectroscopic analysis of combat-related heterotopic ossification.</a> Bone. 57 (2), 335-342 (2013).</li>
</ol>

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.

在目前的研究中，该协议被描述为创伤此前公布的小鼠模型/刻录诱导HO 14-17这涉及创建并发局部肌肉骨骼损伤与跟腱切断术和全球炎症侮辱与部分厚度烧伤。这导致在切断术部位可遵循与串行成像可靠的形成反应骨。迄今为止，所有的小鼠（50个以上）已持续两个跟腱切断术和烧伤已经开发HO可测量的量在切断术部位。量化异位骨的形成，一个校准图像的μCT协议用于在每个期望的时间点。...

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.

异位骨化直接鼠标创伤/烧伤模型

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

Research

JoVE Journal

Medicine

10.1K 次观看.  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.

视频: 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：急性周围组织的创伤模型

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

科研

医学

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.

胫骨肉瘤细胞注射生成原位骨肉瘤和肺转移小鼠模型

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

癌症研究

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.

小鼠牵张成骨模型

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

发育生物学

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.

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

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.

一个简单的临界尺寸股骨缺损模型小鼠

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 ...

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.

骨科手术的小鼠模型研究术后认知功能障碍与组织再生

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

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.

机械转导驱动的类人增生性瘢痕形成小鼠模型

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

生物工程

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

<meta charset="utf8"></head><body>胫骨骨折-针模型：骨科损伤的临床相关小鼠模型

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

胫骨骨折-针模型：骨科损伤的临床相关小鼠模型

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

神经科学

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.

用异体皮肤移植重建的烧伤伤口的穆林模型

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

异位骨化直接鼠标创伤/烧伤模型

摘要

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