Name: transverse fracture of the mouse femur with stabilizing pin
Uploaded: 2021-12-29T13:30:00
Description: Watch this Scientific Journal Video about Transverse Fracture of the Mouse Femur with Stabilizing Pin at JoVE.com

我们感谢Vicki Rosen博士对该项目的财政支持和指导。我们还要感谢哈佛医学院的兽医和IACUC工作人员就无菌技术，动物福祉以及用于开发该协议的材料进行咨询。

所有描述的动物实验都得到了哈佛医学区机构动物护理和使用委员会的批准。该方案使用12周龄的C57BL / 6J小鼠（雄性和雌性）。C57BL / 6J雄性和雌性小鼠在12周龄左右达到峰值骨量，股骨宽度足以适合稳定针，使它们成为用于该方案的适当菌株15。
1. 手术准备
<ol><li>高压灭菌手术设备，包括手术剪刀、直钳、弯曲镊子、手术夹具和金刚石切割轮?...

<ol>
	<li>Black, D. M., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmenopausal+osteoporosis.">Postmenopausal osteoporosis.</a> The New England Journal of Medicine. 374, 2096-2097 (2016).</li><li>Curtis, E. M., Moon, R. J., Harvey, N. C., Cooper, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+fragility+fracture+and+approaches+to+osteoporosis+risk+assessment+worldwide.">The impact of fragility fracture and approaches to osteoporosis risk assessment worldwide.</a> Bone. 104, 29-38 (2017).</li><li>Laurent, M. R., Dedeyne, L., Dupont, J., Mellaerts, B., Dejaeger, M., Gielen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+bone+loss+and+sarcopenia+in+men.">Age-related bone loss and sarcopenia in men.</a> Maturitas. 122, 51-56 (2019).</li><li>NOF - Just for men. National Osteoporosis Foundation Available from: <a target="_blank" href="https://cdn.nof.org/wp-content/uploads/2015/12/Osteoporosis-Fast-Facts.pdf">https://cdn.nof.org/wp-content/uploads/2015/12/Osteoporosis-Fast-Facts.pdf</a> (2019)</li><li>Williams, S. 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=Economic+burden+of+osteoporotic+fractures+in+US+managed+care+enrollees.">Economic burden of osteoporotic fractures in US managed care enrollees.</a> The American Journal of Managed Care. 26, 142-149 (2020).</li><li>Sheen, J. R., Garla, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fracture+healing+overview.">Fracture healing overview.</a> StatPearls. , (2021).</li><li>Holmes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closing+the+gap.">Closing the gap.</a> Nature. 550, 194-195 (2017).</li><li>Duchamp de Lageneste, 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=Periosteum+contains+skeletal+stem+cells+with+high+bone+regenerative+potential+controlled+by+Periostin.">Periosteum contains skeletal stem cells with high bone regenerative potential controlled by Periostin.</a> Nature Communications. 9, 773 (2018).</li><li>Bahney, C. S., 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=Cellular+biology+of+fracture+healing.">Cellular biology of fracture healing.</a> Journal of Orthopaedic Research. 37, 35-50 (2019).</li><li>Li, Z., 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=Fracture+repair+requires+TrkA+signaling+by+skeletal+sensory+nerves.">Fracture repair requires TrkA signaling by skeletal sensory nerves.</a> Journal of Clinical Investigation. 129, 5137-5150 (2019).</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>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+bone.">Production of a standard closed fracture in laboratory animal bone.</a> Journal of Orthopaedic Research. 2, 97-101 (1984).</li><li>Hu, K., Olsen, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblast-derived+VEGF+regulates+osteoblast+differentiation+and+bone+formation+during+bone+repair.">Osteoblast-derived VEGF regulates osteoblast differentiation and bone formation during bone repair.</a> Journal of Clinical Investigation. 126, 509-526 (2016).</li><li>Collier, C. D., 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=Characterization+of+a+reproducible+model+of+fracture+healing+in+mice+using+an+open+femoral+osteotomy.">Characterization of a reproducible model of fracture healing in mice using an open femoral osteotomy.</a> Bone Reports. 12, 100250 (2020).</li><li>Glatt, V., Canalis, E., Stadmeyer, L., Bouxsein, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+changes+in+trabecular+architecture+differ+in+female+and+male+C57BL/6J+mice.">Age-related changes in trabecular architecture differ in female and male C57BL/6J mice.</a> Journal of Bone and Mineral Research. 22, 1197-1207 (2007).</li><li>Garcia, P., 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=A+new+technique+for+internal+fixation+of+femoral+fractures+in+mice:+impact+of+stability+on+fracture+healing.">A new technique for internal fixation of femoral fractures in mice: impact of stability on fracture healing.</a> Journal of Biomechanics. 41, 1689-1696 (2008).</li><li>Holstein, J. H., 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=Advances+in+the+establishment+of+defined+mouse+models+for+the+study+of+fracture+healing+and+bone+regeneration.">Advances in the establishment of defined mouse models for the study of fracture healing and bone regeneration.</a> Journal of Orthopaedic Trauma. 23, 31-38 (2009).</li><li>Garcia, P., 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=Rodent+animal+models+of+delayed+bone+healing+and+non-union+formation:+a+comprehensive+review.">Rodent animal models of delayed bone healing and non-union formation: a comprehensive review.</a> European Cells &amp; Materials. 26 (1-12), 12-14 (2013).</li><li>Histing, T., 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=Ex+vivo+analysis+of+rotational+stiffness+of+different+osteosynthesis+techniques+in+mouse+femur+fracture.">Ex vivo analysis of rotational stiffness of different osteosynthesis techniques in mouse femur fracture.</a> Journal of Orthopaedic Research. 27, 1152-1156 (2009).</li><li>Williams, J. N., Li, Y., Valiya Kambrath, A., Sankar, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Generation+of+closed+femoral+fractures+in+mice:+A+model+to+study+bone+healing.">The Generation of closed femoral fractures in mice: A model to study bone healing.</a> Journal of Visualized Experiments. (138), e58122 (2018).</li><li>Haffner-Luntzer, 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=A+novel+mouse+model+to+study+fracture+healing+of+the+proximal+femur.">A novel mouse model to study fracture healing of the proximal femur.</a> Journal of Orthopaedic Research. 38, 2131-2138 (2020).</li></ol>

该协议中详述的损伤模型包括在自发性骨折愈合期间观察到的所有四个重要步骤，包括（1）随着血肿的形成而引起的促炎反应，（2）骨骼祖细胞从骨膜中募集形成软愈伤组织，（3）成骨细胞矿化愈伤组织，以及（4）破骨细胞重塑骨骼。
本手稿中描述的外科手术针对至少12周大的成年小鼠进行了优化。27 G x 1 1/4 （0.4 mm x 30 mm） 针头用作稳定针，因为它是这个年龄段骨髓腔宽?...

骨折，骨表面连续性的断裂，发生在人群的所有部分。由于衰老或疾病而骨骼脆弱的人，它们变得严重，脆性骨折的医疗保健费用预计将在5年内超过250亿美元1，2，3，4，5。了解骨折修复所涉及的生物学机制将是开发旨在增强愈合过程的新疗法的起点。以前的研究表明，骨折后，会发生四个重要步骤，使骨骼愈合：（1）血肿的形成;（2）形成纤维软骨愈伤组织;（3）软愈伤组织矿化形成骨骼;（4）愈合骨的重塑6，7。许多生物过程被激活以成功愈合骨折。首先，在骨折后立即启动急性促炎反应6，7。然后，骨膜被激活并扩张，骨膜细胞分化成软骨细胞以形成软骨愈伤组织，其生长以填充被破坏的骨节段6，7，8，9留下的间隙。神经和血管细胞侵入新形成的愈伤组织以提供促进修复所需的额外细胞和信号分子6，7，8，9，10。除了有助于愈伤组织形成外，骨膜细胞还分化成成成骨细胞，在桥接愈伤组织中铺设编织骨。最后，破骨细胞重塑新形成的骨骼以恢复到其原始形状和层状结构7，8，9，10，11。许多小组开发了骨折修复的小鼠模型。小鼠早期和最常用的骨折模型之一是Einhorn方法，其中重量从特定的高度12下降到腿上。缺乏对角度和用于诱导骨折的力的控制，导致骨不连续性的位置和大小产生很多可变性。随后，它导致观察到的特定骨折愈合反应的变化。其他流行的方法是手术干预以产生胫骨单皮质缺损或应力性骨折，这些手术诱导相对温和的愈合反应10，13。这些模型中的可变性主要是由于执行该过程的人14。
在这里，详细的小鼠股骨损伤模型允许控制断裂，以提供可重复的损伤，并允许对股骨骨折修复进行定量和定性评估。具体而言，在成年小鼠的股骨中引入完全突破并稳定骨折末端，以解释物理负荷在骨骼愈合中的作用。还详细介绍了使用组织学和显微计算断层扫描（microCT）采集组织和成像愈合过程不同步骤的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>23 G x 1 TW IM (0.6 mm x 2 5mm) needle</td>
			<td>BD precision</td>
			<td>305193</td>
			<td>Use as guide needle</td>
		</tr>
		<tr>
			<td>27 G x 1 &#188; (0.4 mm x 30 mm)</td>
			<td>BD precision</td>
			<td>305136</td>
			<td>Use as stabilizing pin</td>
		</tr>
		<tr>
			<td>9 mm wound autoclip applier/remover/clips kit</td>
			<td>Braintree Scientific, INC</td>
			<td>ACS-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8 GX</td>
			<td>Electron Microscopy Sciences</td>
			<td>10350</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Millipore Sigma</td>
			<td>AX1303</td>
			<td></td>
		</tr>
		<tr>
			<td>Circular blade X926.7 THIN-FLEX</td>
			<td>Abrasive technologies</td>
			<td>CELBTFSG633</td>
			<td></td>
		</tr>
		<tr>
			<td>DREMEL 7700-1/15, 7.2 V Rotary Tool Kit</td>
			<td>Dremel</td>
			<td>7700 1/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>ThermoScientific</td>
			<td>7111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine curved dissecting forceps</td>
			<td>VWR</td>
			<td>82027-406</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxulin Gill 2</td>
			<td>Sigma-Aldrich</td>
			<td>GHS216</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Millipore Sigma</td>
			<td>HX0603-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical kit</td>
			<td>VWR</td>
			<td>95042-540</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma-Aldrich</td>
			<td>1625</td>
			<td></td>
		</tr>
		<tr>
			<td>Phloxine B</td>
			<td>Sigma-Aldrich</td>
			<td>P4030</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine Swabs</td>
			<td>PDI</td>
			<td>S23125</td>
			<td></td>
		</tr>
		<tr>
			<td>SCANCO Medical &#181;CT35</td>
			<td>Scanco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slow-release buprenorphine</td>
			<td>Zoopharm</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

transverse fracture of the mouse femur with stabilizing pin

骨折修复是骨骼的基本功能，无法在 体外可靠地建模。小鼠损伤模型是测试基因，基因产物或药物是否影响骨修复的有效方法，因为小鼠骨骼概括了在人类骨折愈合期间观察到的阶段。当小鼠或人类骨折时，炎症反应被启动，骨膜，围绕骨骼本身的干细胞生态位，被激活并扩张。然后，居住在骨膜中的细胞分化形成血管化的软愈伤组织。从软愈伤组织到硬愈伤组织的转变发生在新募集的骨骼祖细胞分化成矿化细胞时，骨折末端的桥接导致骨愈合。然后，矿化的愈伤组织进行重塑，以恢复愈合骨的原始形状和结构。已经使用各种损伤模型在小鼠中研究了骨折愈合。尽管如此，概括整个生物过程的最佳方法是突破包含两个皮质的长骨的横截面。该协议描述了如何安全地进行稳定的横贯性股骨骨折以评估成年小鼠的愈合情况。还提供了手术方案，包括详细的收获和成像技术，以表征骨折愈合的不同阶段。

在C57BL / 6J小鼠中，成功的手术完成了前面提到的愈合步骤，几乎没有局部炎症反应或假手术对侧股骨的骨膜受累。术后几小时形成血肿，骨膜被激活以招募骨骼祖细胞进行软骨发育。各种细胞群，如Prx1 + 间充质祖细胞，可以在修复过程中使用市售荧光报告小鼠模型进行追踪（图3）。在骨折后5天（dpf），Alcian Blue染色可用于可视化软愈伤组织，随后量化软骨区域（<s...

Watch this Scientific Journal Video about Transverse Fracture of the Mouse Femur with Stabilizing Pin at JoVE.com

Transverse Fracture of the Mouse Femur with Stabilizing Pin

该协议描述了一种对成年小鼠进行骨折并监测愈合过程的方法。

带稳定针的小鼠股骨横断

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

该协议描述了一种在成年小鼠身上进行和研究股骨骨折的可靠技术。这种技术简单，快速，并且需要执行的工具最少。它允许表征骨折愈合的不同阶段。个体最初可能难以正确对齐骨折末端并在小鼠之间产生一致的骨折。密切关注骨折部位，插入针和闭合伤口将提高该手术的成功率。首先，通过皮下注射每公斤丁丙诺啡0.05毫克来制备动物。使用电动修剪器在两条大腿上剃除两乘两厘米的正方形，对应于股骨的位置。使用碘对剃须区域进行灭菌，并用70%乙醇溶液冲洗。做一个五毫米的切口，露出下面的筋膜。通过切割筋膜暴露覆盖股骨的肌肉。在不损伤组织的情况下将肌肉与股骨分开，然后将肌肉与骨骼分离并固定股骨以进行切割。在股骨轴中间进行横向切口。将导针插入骨髓腔的远端部分，并将线推过膝关节。然后，取出导针并从近端执行类似的程序。将针头留在近端，其尖端出现在皮肤上方。在导针的尖端内插入一个稳定销，推动它，使其在导针被拉出时进入近端腔，然后丢弃导针。将远端与近端对齐，使螺纹在离开膝关节时穿过远端骨髓腔。使用手术钳拉动针尖，使近端和远端部分拉在一起。然后将手术针的末端朝向骨折部位折叠，以重新对齐骨折末端。使用线切割器从针的底部取出塑料。扭转针脚的末端，以避免任何内部组织损伤。最后，将肌肉置换到股骨上，并用夹子捏合皮肤来关闭伤口开口。执行类似的程序，另一条腿上没有股骨骨折并闭合伤口。手术成功后，荧光成像揭示了在股骨骨折修复过程中发现的各种Messent-Kinal祖细胞，例如Prx1阳性骨膜细胞。骨折股骨的阿尔西安蓝染色揭示了软愈伤组织的形成。此外，还确定了愈伤组织或软骨形成的百分比。在断裂后约28 d采用Micro-CT，探测到矿化过程以及愈伤组织间隙和断裂间隙。还观察到，如果骨折末端未正确对齐或未用销钉固定，则裂缝末端的所有或一侧均没有无情形成。当尝试这个程序时，请记住小心地对齐断股骨的两端，并用针固定它们，以确保适当的愈合。手术后，可以收集股骨进行成像和分子分析。

Research

JoVE Journal

Biology

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

科研

生物学

Transverse Aortic Constriction in Mice

在小鼠体内的横向主动脉缩窄

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

表征隔离，通风，和仪表小鼠肺灌注与脉动流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

冷冻断裂/冷冻蚀刻的生物电子显微技术的基本要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

表型鼠标肺功能<em&gt;在体内</em&gt;与肺弥散功能

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

用荧光线粒体分离条件永生化的小鼠肾小球内皮细胞

Glomerular endothelial cell (GEC) dysfunction can initiate and contribute to glomerular filtration barrier breakdown. Increased mitochondrial oxidative ...