异位骨化直接鼠标创伤/烧伤模型

摘要

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.

摘要

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.

引言

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

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研究方案

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。

1. 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。
2. 辖丁丙诺啡，0.1毫克/千克，皮下之前立即手术镇痛。
3. 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。
4. 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。
5. 准备手术部位，包括后爪来使用3-交替碘伏磨砂膝盖。
6. 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。
7. 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。
8. 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。
9. 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。
10. 干烧伤部位用纱布和应用TEGADERM敷料。
11. 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。
12. 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。

2.μCT采集与分析

1. 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。
2. 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
3. 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。
4. 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。
5. 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。
6. 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。

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结果

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。...

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讨论

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...

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材料

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