这份出版物中的研究报告得到了国家卫生研究院的 F30AR071201 和 R01AR066028 的全国关节炎和肌肉骨骼和皮肤病研究所的支持。

这项议定书的制定是为了确保动物不被不必要地使用, 并幸免于一切不必要的痛苦和痛苦;它遵守所有适用的联邦、州、地方和机构的法律和指导动物研究的准则。该议定书是在一个由实验室动物医学专业兽医指导的大学范围内的实验动物医学计划的指引下制定的。该议定书得到了机构动物保育和使用委员会 (IACUC) 的审查和批准。
1. 断裂塔施工
注: 所有部件都列在材料部分 (材料表) 中。在补充图 1-12中, 为加工和3维印刷零件提供了详细的技术图纸。组件技术图纸包括所有安装部件的紧固件详细信息 (辅助图1、2、7和9)。
<ol><li>
支持子组件 注: 有关支持子组件的技术图, 请参阅辅助图 1。<ol>
<li>将光束支撑--横梁支撑中点的颚部- -水平截面。</li>
		<li>附加光束支撑- -垂直 1到光束支撑的顶面--颚部, 2 从光束支撑--水平剖面。</li>
		<li>将光束支撑--垂直 2到横梁支撑的顶面--中点的水平剖面(从末端 7)。</li>
		<li>连接梁, 支撑板安装到两端的横梁支架--垂直 1和横梁支撑--垂直 2。板支撑的端面应与横梁支承的背面齐平--垂直 2。</li>
	</ol>
</li>
	<li>
Ram 组件 注: 有关 ram 组件的技术图纸, 请参阅辅助图 2。<ol>
<li>机器的块停止和块指南(补充图 3);杆 Ram (辅助图 4);螺钉对准(辅助图 5);和板材安装(补充图 6)。</li>
		<li>将平板安装到横梁支架上--支撑子组件的板安装。</li>
		<li>按以下顺序滑动第一线性套筒轴承;块指南;第二线性套筒轴承;在拉杆 Ram上停止阻塞。将导轨和块连接到板上安装。</li>
		<li>将三⅜螺母连接到拉杆 Ram的螺纹部分。一个应该与杆的末端冲洗与电磁铁接触。另外2将用于调整裂缝深度。</li>
		<li>将拉杆 Ram中的小树林对准前方, 并将对准螺钉插入块导轨的螺纹孔中。</li>
	</ol>
</li>
	<li>
磁铁组件 注: 对于磁铁子组件的技术图纸, 请参阅辅助图 7。<ol>
<li>焊料电磁铁导致导线 (极性不是一个因素为电磁铁操作)。允许足够长的时间到达地板, 在那里骨折装置将被定位。使用拉链或另一种形式的附加应力减轻电线。</li>
		<li>剥离电源的末端, 并将其连接到脚踏板。最后, 将导线连接到脚踏板上的 "关闭" (通常是打开的) 配置。测试电路, 以确保电磁铁是在脚开关没有按下。这将使 ram 在骨折前保持。</li>
		<li>使用添加剂制造设备打印安装磁体(辅助图 8A和8B), 或从铝加工零件。</li>
		<li>将电磁铁连接到安装磁铁上。</li>
		<li>将 2角括号附加到光束支架上-磁铁。</li>
		<li>按以下顺序, 将拉杆磁铁穿过上角支架, 加入一个¼螺母;安装磁铁;两个¼的坚果;和底部角括号。在每个端上用两个¼螺母保护组件。</li>
	</ol>
</li>
	<li>
完整的组件 注: 有关完整组件的技术图纸, 请参阅辅助图 9。<ol>
<li>将磁体组件连接到横梁的顶面, 支撑板安装。</li>
		<li>调整光束支撑的对准-磁铁, 使磁铁与杆, Ram。 注: 如果杆不释放, 当踏板被按下, 减少接触面积之间的电磁铁和杆通过移动光束支持-磁铁。</li>
		<li>机器支架腿颚(辅助图 10)。</li>
		<li>将两个支架腿颚连接到横梁支架--颚部。当掉落时, ram 的尖端应该与每个颚的距离相等。</li>
		<li>将平台骨折(补充图 11A和11B) 置于颌骨上方。</li>
		<li>使用添加剂制造设备打印夹具定位断裂(辅助图 12A和12B) 和夹具销规(补充图 13A和13B), 或机器零件从铝。 注:夹具的尺寸将按步骤2中详细说明的优化步骤计算。</li>
		<li>将夹具定位断裂连接到平台断裂。</li>
		<li>确认撞击深度可以使用杆 Ram上的两个停止螺母进行调整。</li>
		<li>确认撞击速度可以通过上下移动安装磁体来调整。</li>
		<li>确认骨折的宽度可以通过移动方腿颚靠近或远离杆 Ram来调整。</li>
	</ol>
</li>
</ol>2. 断裂优化
<ol><li>断裂位置
	<ol>
<li>获得5例安乐死动物的代表性样品中骨折的肢体 (股骨或胫骨) 的影像。 注: 样品应与标本相匹配, 该样本将用于基于年龄、基因型和性别的实验性协议。即使最后的协议只要求一个骨折的肢体, 两个样本四肢将使用。</li>
		<li>将肢体切向 x 射线束, 以获得真实的侧向和前/后视图的骨骼。将已知维度的对象放置在成像平面上, 以提供用于分析的刻度。</li>
		<li>注: 如影像股骨, 确保肢体完全伸展, 股骨与胫骨在同一轴向平面。</li>
		<li>在要断开的肢体的射线照相上标记所需的骨折位置 (图 1条虚线)。测量从跟骨关节到明显骨折的水平 (图 1A)。计算所有试件的平均断裂长度 (FL)。股骨髁间凹槽的测量。</li>
	</ol></li>
	<li>断裂定位夹具
	<ol>
<li>测量从一个支撑铁砧的外部表面到断头台撞击 (CGI) 中心的距离 (图 2)。将CGI从FL中减去, 在步骤2.1.4 中描述, 以计算断裂定位夹具深度 (JD)。机器或 3 d-打印 U 形通道, 其高度和宽度等于铁砧, 深度等于JD (图 3a)。辅助图 12A和12B中包含了一个示例技术图纸和 CAD 文件。 注意: 当肢体被放置在夹具中时, 脚的背部应该与断头台撞击的表面距离最远。如果肢体需要额外的间隙, 请修改 U 形通道。</li>
		<li>将标本放置在骨折器械中, 用于股骨骨折或仰卧位胫骨骨折 (图 4)。将脚背压在骨折定位夹具的末端。手动压低断头台直到肢体骨折。获得骨折的肢体的射线照相, 以确认夹具大小和骨折位置 (图 2B)。</li>
		<li>增加jd如果骨折位置太远端骨, 或减少jd , 如果骨折位置太近骨。</li>
	</ol></li>
	<li>引脚参数的镇定
	<ol>
<li>
引脚长度:使用在步骤2.1 中获得的射线照相, 测量四肢长度(从胫骨高原到后踝的水平, 胫骨骨折, 或髁间凹槽到较大的转子股骨骨折。将骨骼长度乘以0.9 以计算 pin 长度 (PL) (图 1A和3B)。</li>
		<li>
针宽:使用步骤2.1 中获得的射线照相, 测量骨折肢体的最小髓质直径 (MD) (图 1A)。选择一针, 其尺寸大约相当于髓质直径, 长度超过 1.5 x PL。 注:14 周大的 C57BL/6J 鼠的大约针大小分别为股骨和胫骨的22克、1½和27克, 1¼。</li>
	</ol></li>
	<li>销切量规
	<ol>
<li>2.4.1 机器或 3 d 打印一个长度等于PL减去针长 (CGL) 的量规 (图 3B;补充图 13A及13B)。一端应该有一个悬臂, 以休息对针的中心, 另一个应该指出在哪里应该削减 pin。辅助图 13A和13B中包含了一个示例技术图纸和 CAD 文件。</li>
	</ol></li>
	<li>髓内针骨折稳定化
	<ol>
<li>使用步骤2.1 中的非骨折试验标本, 从胫骨中到股骨中移除毛发, 用电动剪或脱毛膏将头发取出, 露出膝关节。</li>
		<li>
胫骨钉扎: 插入针经皮侧的髌骨韧带。收回髌韧带内侧, 将针尖与胫骨轴对齐。使用扩孔运动, 轻轻地突破胫骨平台, 引导针下髓腔。</li>
		<li>
股骨钉: 插入针经皮侧的髌骨韧带。收回髌骨韧带内侧, 并将针尖与股骨髁间凹槽的轴线对齐。采用扩孔运动, 轻轻地突破髁间凹槽的关节面, 引导针下髓腔。</li>
		<li>使用步骤2.4 中制造的量规, 直到暴露的针等于测量长度。收回针提供足够的空间 (~ 3 毫米), 以削减针在水平指示的测量。 注: 一定要在切割时保持针的近端 (塑料) 末端, 所以它不会变成危险的弹丸。</li>
		<li>使用针刀将销的远端的0.3 毫米卷曲, 然后在量规的水平上切割针脚。使用直径大于针直径1.5x 的杆将引脚固定在关节表面。 注: 通过增加针骨接触, 压接可防止针的旋转和迁移。</li>
		<li>获得射线照相以确认针延长肢髓质管的长度, 不从近端或远端凸出 (图 1C)。</li>
	</ol></li>
	<li>冲击深度
	<ol>
<li>使用步骤2.1 中获得的射线照相, 测量所需骨折水平的皮层直径 (图 1A)。计算所有试验标本的平均皮质直径 (CD)。</li>
		<li>用步骤2.2 中制造的断裂定位夹具将骨折装置中步骤2.5 中的固定试件定位。将撞击 ram 放在未受伤的肢体上。 注意: 不要让 ram 掉下来;在这个优化步骤中, 骨骼应该保持完好。</li>
		<li>在 ram 上施加足够的向下力来压缩软组织, 但不能骨折骨。将撞击深度 (ID) 调整为 0.75 x CD (图 2)。 注: 理想的撞击深度为 0.5 x CD , 当骨折时没有任何软组织。使用0.75 帐户进行额外的软组织压缩。</li>
	</ol></li>
	<li>砧宽
	<ol>
<li>为小鼠胫骨或股骨设置砧宽 (AW) 为0.4 厘米 (图 2)。 注意: 建议更宽的宽度, 如大鼠等较大的标本。</li>
	</ol></li>
	<li>Ram 重量
	<ol>
<li>建议对小鼠标本的最小重量为250克。 注意: 额外的重量可以被螺纹到 ram 为更大的标本 (图 2)。</li>
	</ol></li>
	<li>冲击速度
	<ol>
<li>将放置高度 (DH) 设置为2厘米 (图 2)。将 ram 放置在其起始位置, 将其连接到活化电磁铁。</li>
		<li>在骨折装置中放置一个试肢。将脚背按在步骤2.2 中制造的骨折定位夹具上。简要地压低脚踏开关释放 ram, 然后将其重置为其起始位置。</li>
		<li>对受影响的试验肢体进行射线照相。分析肢体骨折的任何证据 (图 1D)。 注意: 当使用具有受控冲击深度的低速速度时, 这可能是微妙的。</li>
		<li>如果未生成任何断开, 请重复步骤 2.9.1 2.9.3, 并将放置高度增加2厘米。</li>
		<li>如果生成断开, 则记录下拉高度, 并将其乘以1.1。这是新生署。</li>
		<li>使用DH从步骤 2.9.5, 骨折的下一个试验肢体。</li>
		<li>如果未生成任何断开, 请重复步骤 2.9.1 2.9.6, 并将放置高度增加2厘米。</li>
		<li>如果生成了断开, 请重复步骤 2.9.6 2.9.7, 直到使用所有测试示例。从优化中记录最终的DH和所有参数 (FL、 CGI、 JD、 PL、 MD、 PS、 CGL、 CD、 ID、 AW和cd-rw)。记录试验标本的年龄、性别、基因型和体重。</li>
	</ol></li>
</ol>3. 闭合稳定的断裂世代
<ol><li>
设置
	<ol>
<li>通过高压釜、热珠浸泡或其等效物灭菌所有设备和仪器。</li>
		<li>将加热元件放在手术台上, 并将其设置为最佳温度。用外科悬垂盖住这个元素。在2的外科悬垂中准备 3 x 3, 在中间切开一个0.75 英寸的圆圈。</li>
		<li>在每次试验前确认裂缝塔的调整 (图 2)。将ID、 AW、 cd-rw和DH设置为要研究的样本中特定于性别、年龄和基因型的优化协议中的值。</li>
		<li>权衡和记录动物的重量。</li>
	</ol>
</li>
	<li>
手术
	<ol>
<li>用吸入麻醉药 (异氟醚: 4-5% 进行诱导; 1-2% 用于维护) 或另一种已建立的实验室麻醉协议, 使鼠标充分镇静。呼吸率应为 55-100 呼吸/分钟。动物不应该对后肢脚趾捏起反应。</li>
		<li>管理术后镇痛的第一剂量丁丙诺啡 (0.1 毫克/千克皮下)。</li>
		<li>应用眼部润滑, 防止角膜干燥。</li>
		<li>用电动剪刀从胫骨中到股骨中移除动物的毛发, 露出膝盖关节。使用无反应胶带清洁多余毛发的部位。用70% 乙醇湿拭子准备固定部位。必要时重复清除切口区域的所有毛发。</li>
		<li>用聚维酮碘和70% 乙醇的替代拭子制备和清洁固定区域。使用两个替代拭子序列, 以确保不孕。</li>
		<li>在皮肤经过适当消毒后, 在手术部位放置一个悬垂。</li>
		<li>使用步骤2.5 中描述的协议将肢体折断。获得射线照相, 以确认针延长髓质管长度, 但不突出从近端或远端。</li>
		<li>打开电磁铁并连接撞击 ram 将其放置在起始位置。</li>
		<li>将标本放置在骨折器械中, 将其置于股骨骨折或仰卧位胫骨骨折的俯卧位。固定的肢体应放置在铁砧和在骨折定位夹具与背部的脚压在夹具外。</li>
		<li>当一只手按下脚, 并确保只有肢体在撞击 ram 目标区域, 简要地压低脚踏开关释放 ram。替换起始位置的 ram。</li>
		<li>获取射线照相并确认骨折位置和类型。</li>
	</ol>
</li>
	<li>
术后管理
	<ol>
<li>监测动物每15分钟从麻醉恢复, 直到动物意识, 可以保持胸骨卧床, 是动态的。确认动物能在72小时内走动。</li>
		<li>将动物单独饲养, 直到完全恢复。</li>
		<li>在48小时内保持镇痛, 每12小时服用丁丙诺啡 (0.1 毫克/千克)。</li>
		<li>监测和记录动物的健康状况每日 7-10 d 或直到安乐死。</li>
	</ol>
</li>
	<li>
断裂后分析
	<ol>
<li>测量FL、 PL、 CD、 MD和断裂模式。记录主数据文件中的度量值。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Corso, P., Finkelstein, E., Miller, T., Fiebelkorn, I., Zaloshnja, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+lifetime+costs+of+injuries+in+the+United+States.">Incidence and lifetime costs of injuries in the United States.</a> Injury Prevention. 12 (4), 212-218 (2006).</li><li>Nguyen, N. D., Ahlborg, H. G., Center, J. R., Eisman, J. A., Nguyen, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+lifetime+risk+of+fractures+in+women+and+men.">Residual lifetime risk of fractures in women and men.</a> Journal of Bone and Mineral Research: The Official Journal of the American Society for Bone and Mineral Research. 22 (6), 781-788 (2007).</li><li>Thompson, Z., Miclau, T., Hu, D., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+intramembranous+ossification+during+fracture+healing.">A model for intramembranous ossification during fracture healing.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 20 (5), 1091-1098 (2002).</li><li>Cheung, K. M. C., Kaluarachi, K., Andrew, G., Lu, W., Chan, D., Cheah, K. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> Journal of Orthopaedic Research. 21 (4), 685-690 (2003).</li><li>Connolly, C. K., 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+reliable+externally+fixated+murine+femoral+fracture+model+that+accounts+for+variation+in+movement+between+animals.">A reliable externally fixated murine femoral fracture model that accounts for variation in movement between animals.</a> Journal of Orthopaedic Research. 21 (5), 843-849 (2003).</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=An+internal+locking+plate+to+study+intramembranous+bone+healing+in+a+mouse+femur+fracture+model.">An internal locking plate to study intramembranous bone healing in a mouse femur fracture model.</a> Journal of Orthopaedic Research. 28 (3), 397-402 (2010).</li><li>Gr&#246;ngr&#246;ft, I., 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=Fixation+compliance+in+a+mouse+osteotomy+model+induces+two+different+processes+of+bone+healing+but+does+not+lead+to+delayed+union.">Fixation compliance in a mouse osteotomy model induces two different processes of bone healing but does not lead to delayed union.</a> Journal of Biomechanics. 42 (13), 2089-2096 (2009).</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 (1), 97-101 (1984).</li><li>Huang, C., 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=The+spatiotemporal+role+of+COX-2+in+osteogenic+and+chondrogenic+differentiation+of+periosteum-derived+mesenchymal+progenitors+in+fracture+repair.">The spatiotemporal role of COX-2 in osteogenic and chondrogenic differentiation of periosteum-derived mesenchymal progenitors in fracture repair.</a> PloS One. 9 (7), 100079 (2014).</li><li>Waki, 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=Profiling+microRNA+expression+during+fracture+healing.">Profiling microRNA expression during fracture healing.</a> BMC Musculoskeletal Disorders. 17, 83 (2016).</li><li>Yee, 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=Sclerostin+antibody+treatment+improves+fracture+outcomes+in+a+Type+I+diabetic+mouse.">Sclerostin antibody treatment improves fracture outcomes in a Type I diabetic mouse.</a> Bone. 82, 122-134 (2016).</li><li>Wong, E., 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+low-molecular-weight+compound+enhances+ectopic+bone+formation+and+fracture+repair.">A novel low-molecular-weight compound enhances ectopic bone formation and fracture repair.</a> The Journal of Bone and Joint Surgery. American Volume. 95 (5), 454-461 (2013).</li><li>Prodinger, P. 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=Does+Anticoagulant+Medication+Alter+Fracture-Healing?+A+Morphological+and+Biomechanical+Evaluation+of+the+Possible+Effects+of+Rivaroxaban+and+Enoxaparin+Using+a+Rat+Closed+Fracture+Model.">Does Anticoagulant Medication Alter Fracture-Healing? A Morphological and Biomechanical Evaluation of the Possible Effects of Rivaroxaban and Enoxaparin Using a Rat Closed Fracture Model.</a> PloS One. 11 (7), 0159669 (2016).</li><li>Menzdorf, 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=Local+pamidronate+influences+fracture+healing+in+a+rodent+femur+fracture+model:+an+experimental+study.">Local pamidronate influences fracture healing in a rodent femur fracture model: an experimental study.</a> BMC Musculoskeletal Disorders. 17, 255 (2016).</li><li>Hagiwara, Y., 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=Fixation+stability+dictates+the+differentiation+pathway+of+periosteal+progenitor+cells+in+fracture+repair.">Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.</a> Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 33 (7), 948-956 (2015).</li><li>Gardner, M. J., 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=Differential+fracture+healing+resulting+from+fixation+stiffness+variability:+a+mouse+model.">Differential fracture healing resulting from fixation stiffness variability: a mouse model.</a> Journal of Orthopaedic Science: Official Journal of the Japanese Orthopaedic Association. 16 (3), 298-303 (2011).</li><li>Catma, M. F., 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=Remote+ischemic+preconditioning+enhances+fracture+healing.">Remote ischemic preconditioning enhances fracture healing.</a> Journal of Orthopaedics. 12 (4), 168-173 (2015).</li><li>Lichte, 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=Impaired+Fracture+Healing+after+Hemorrhagic+Shock.">Impaired Fracture Healing after Hemorrhagic Shock.</a> Mediators of Inflammation. 2015, 132451 (2015).</li><li>Lopas, L. 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=Fractures+in+geriatric+mice+show+decreased+callus+expansion+and+bone+volume.">Fractures in geriatric mice show decreased callus expansion and bone volume.</a> Clinical Orthopaedics and Related Research. 472 (11), 3523-3532 (2014).</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 (1), 97-101 (1984).</li><li>Marturano, J. E., 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=An+improved+murine+femur+fracture+device+for+bone+healing+studies.">An improved murine femur fracture device for bone healing studies.</a> Journal of Biomechanics. 41 (6), 1222-1228 (2008).</li><li>Jackson, R. W., Reed, C. A., Israel, J. A., Abou-Keer, F. K., Garside, H. <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+experimental+fracture.">Production of a standard experimental fracture.</a> Canadian Journal of Surgery. Journal Canadien De Chirurgie. 13 (4), 415-420 (1970).</li><li>Byrne, M., Cleveland, B., Marturano, J., Wixted, J., Billiar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+reproducible+murine+femoral+fracture+device.+Conference:+Bioengineering+Conference,+2007.">Design of a reproducible murine femoral fracture device. Conference: Bioengineering Conference, 2007.</a> NEBC '07. IEEE 33rd Annual Northeast. , (2007).</li><li>Carter, D. R., Hayes, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compact+bone+fatigue+damage-I.+Residual+strength+and+stiffness.">Compact bone fatigue damage-I. Residual strength and stiffness.</a> Journal of Biomechanics. 10 (5), 325-337 (1977).</li><li>McGee, A., Qureshi, A., Porter, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biomechanics+and+patterns+of+limb+fractures.">Review of the biomechanics and patterns of limb fractures.</a> Trauma. 6 (1), 29-40 (2004).</li></ol>

作者没有什么可透露的。

这种断裂优化和生成协议为研究人员提供了一种有效的方法来获得骨折参数和执行一个微创程序, 产生精确, 可重复, 横向骨折。此外, 该协议还建立了一组常见的断裂生成参数, 从而促进了研究人员之间的方法一致性。这些参数将使创建一个常见的断开数据库, 根据各种参数 (如年龄、性别、性别和基因型) 建立骨折标准。对断裂变量的优化显著降低了样本异质性-减少了浪费时间、资源丢失?...

骨折愈合的研究对于解决大的临床和经济问题是必要的。每年有1200万多起骨折在美国治疗1, 成本为800亿美元2。男性或女性在其一生中遭受骨折的可能性为25% 和 44%, 分别为3。随着并发症的增加, 随着年龄的增长, 骨折愈合的相关问题将会增加。为了研究和解决这一问题, 需要建立可靠的断裂产生和稳定模型。啮齿类动物模型是理想地适合这个目的。它们提供临床相关性, 可以修改以解决特定的情况 (即多发伤、开放性、闭合性、缺血性和感染性骨折)。除了复制临床情况外, 动物骨折模型对于了解骨骼生物学和开发治疗和设备是很重要的。然而, 试图研究干预之间的差异可能是复杂的变异引入不一致的断裂产生。因此, 在动物模型中产生可重现和持续闭合的骨折, 对于肌肉骨骼研究领域是必不可少的。
尽管通过确保适当的遗传背景、性别、年龄和环境条件, 适当控制潜在的主体异质性, 但临床相关的一致骨损伤的产生是一个重要变量, 影响必须控制的重现性。使用不一致的裂缝进行统计比较, 实验噪声和高变异性4;此外, 骨折变异性可能导致不必要的动物死亡, 因为需要增加样本大小或必要的弄死动物的粉碎或 malpositioned 骨折。本文所述方法的目的是优化特定于样本类型的断裂生成参数, 并产生一致的断裂位置和模式。
目前的断裂产生模型分为两大类, 各有优缺点。开放性骨折 (截骨) 模型进行手术暴露骨, 之后骨折是由切割骨或削弱它, 然后手动打破它5,6,7,8。该方法的优点是裂缝部位的直接可视化, 以及更一致的断裂位置和模式。然而, 损伤途径和机制的生理和临床相关性是有限的。此外, 开放性骨折产生的方法需要手术方法和关闭的长期期间, 啮齿类动物暴露在增加的危险的污染。
封闭技术解决了许多开放技术的局限性。闭合技术产生骨折使用外部应用钝力损伤, 导致损伤的骨骼和周围组织, 更类似于人的临床损伤。最常见的方法是 Bonnarens 和埃霍恩在 1984年9描述。他们描述了一个加权断头台被用来传授钝外伤, 以打破骨骼, 而不造成任何外部皮肤创伤。该方法已广泛应用于研究遗传学 10、11、药理治疗12、13、14、15、力学16的效果,17、生理学18、19、20对小鼠和大鼠骨愈合的影响。闭合方法的好处是生理上相关的断裂, 实验的重现性和严密是受断裂非均质的限制。不一致的断裂产生的结果是有限的组间分化, 失去标本, 并增加动物需要达到统计学意义。
控制断裂产生和稳定的变异性是产生有意义结果的必要条件。为了正确研究骨折修复的生物学, 需要一个简单而健壮的断裂模型。该模型应可翻译为啮齿动物物种, 骨骼类型 (例如股骨或胫骨), 并跨越可变的小鼠遗传背景和诱发突变。此外, 理想的程序应该在技术上简单, 并产生一致的结果。为解决骨折异质性, 本文所描述的方法是构造一个良好的控制骨折装置, 然后可以用来优化参数和产生一致闭合的骨折, 无论年龄, 性别, 或基因型。

<table border="0" cellpadding="0" cellspacing="0" style="width:911px;" width="910">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Support Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td style="width:315px;">Supplementary Figure 1</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Jaw Section&#160;</td>
			<td style="width:209px;">80/20</td>
			<td>1003 x 9.00</td>
			<td>w/ #7042 at A, C, in Left End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Horizontal Section</td>
			<td style="width:209px;">80/20</td>
			<td>1002 x 14.00</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Vertical 1</td>
			<td style="width:209px;">80/20</td>
			<td>1050 x 10.50&#160;</td>
			<td>w/ #7042 at A in Left End and at A in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Vertical 2</td>
			<td style="width:209px;">80/20</td>
			<td>1010 x 10.50&#160;</td>
			<td>w/ #7042 at D, B in Left End and at A in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Plate Mount</td>
			<td style="width:209px;">80/20</td>
			<td>1030 x 8.00&#160;</td>
			<td>w/ #7036 at Left End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Beam, Support--Magnet</td>
			<td style="width:209px;">80/20</td>
			<td>1010 x 13.50&#160;</td>
			<td>w/ #7042 at A, C, in Right End</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Anchors (3)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3392</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Double Anchor (3)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3091</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Bolt Assembly (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3386</td>
			<td>1/4-20 x 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Button Head Socket Cap Screw (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">3604</td>
			<td>1/4-20 x 3/4&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ram Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 2</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Block, Stop</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 3</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Block, Guide</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 3</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Rod, Ram</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 4</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Alignment Screw</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 5</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Plate, Mounting</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 6</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Linear Sleeve Bearing (2)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">8649T2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Hex Nut (3)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>92673A125</td>
			<td>3/8-16 UNC</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (8)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A108</td>
			<td>4/40 x 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (6)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A032</td>
			<td>4/40 x 1 1/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (1)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A267&#160;</td>
			<td>10/32 3/8&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Magnet Subassembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 7</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Mount, Magnet</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 8</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Power Supply</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>70235K23</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Foot Switch</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>7376k2</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Electromagnet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>5698k111</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Wire - 10 feet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">9936k12</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Rod, Magnet</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>95412A566</td>
			<td>1/4&#34; Threaded Rod x 7&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Corner Bracket (6)</td>
			<td style="width:209px;">80/20</td>
			<td style="width:147px;">4108</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Socket Cap Screw (1)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td style="width:147px;">92196A705</td>
			<td>10/32 1 1/4&#34;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Hex Nut (4)</td>
			<td style="width:209px;">McMaster-Carr</td>
			<td>92673A113</td>
			<td>1/4-20 UNC</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Complete Assembly</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 9</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Bracket, Leg Jaw (2)</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 10</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Platform, Fracture</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 11</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Jig, Positioning-Fracture</td>
			<td style="width:209px;">Custom</td>
			<td style="width:147px;"></td>
			<td>Supplementary Figure 12</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Other</td>
			<td style="width:209px;"></td>
			<td style="width:147px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Pin Cutter</td>
			<td style="width:209px;">Medical Supplies and Equipment</td>
			<td style="width:147px;">150S</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:240px;">Needles</td>
			<td style="width:209px;">Sigma</td>
			<td style="width:147px;">Z192430, Z192376&#160;</td>
			<td>23g x 1.5&#34; - mouse femur, 27g x 1.25&#34; - mouse tibia</td>
		</tr>
	</tbody>
</table>

fracture apparatus design and protocol optimization for closed-stabilized fractures in rodents

在动物模型中可靠地生成一致稳定的骨折, 对于了解骨再生生物学和开发治疗方法和装置是必不可少的。然而, 现有的伤害模型受到不一致的困扰, 造成动物和资源的浪费和数据不完善。为了解决断裂异质性的问题, 本文所述方法的目的是优化每种动物的断裂生成参数, 并产生一致的断裂位置和模式。该协议说明了在小鼠菌株之间可能存在的骨骼大小和形态学的变化, 并且可以适应于其他物种 (如老鼠) 产生一致的骨折。此外, 还介绍了一种经济高效、可调节的断裂装置。与目前稳定的断裂技术相比, 优化方案和新的断裂装置表明, 稳定断裂模式和位置的一致性增加。通过使用特定于样本类型的优化参数, 所述协议提高了诱发创伤的精确度, 最大限度地减少了闭合断裂生成过程中通常观察到的断裂异质性。

以前在我们实验室使用的断头台是在2004年开发的, 是基于埃霍恩21出版的模型。该设计不允许调整, 以充分考虑任何差异的骨骼形态学和不允许可重复的定位的肢体。此外, 以前的仪器需要两个人来操作它。因此, 我们设计, 工程, 并建立了一个新的断裂装置。主要设计目标是可能的高保真度调整的裂缝深度, 冲击力, 三点接触, 和动物定位。该设计基于 Martur...

Watch this Scientific Journal Video about Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents at JoVE.com

Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

该协议的目标是优化裂缝产生参数, 以产生一致的裂缝。这项协议解释了动物之间可能存在的骨骼大小和形态学的变化。此外, 还介绍了一种经济高效、可调节的断裂装置。

鼠类闭合稳定骨折的断裂装置设计与协议优化

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing ...

fracture-apparatus-design-protocol-optimization-for-closed-stabilized

Research

JoVE Journal

Medicine

13.3K 次观看.  Michigan State University. 该协议的目标是优化裂缝产生参数, 以产生一致的裂缝。这项协议解释了动物之间可能存在的骨骼大小和形态学的变化。此外, 还介绍了一种经济高效、可调节的断裂装置。

视频: Fracture Apparatus Design and Protocol Optimization for Closed-stabilized Fractures in Rodents

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

的的膀胱测压小老鼠:A膀胱Chemosensation研究的使用

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

科研

医学

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

人体胰腺的收集协议

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

联体小鼠:一个详细的协议

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

特发性脊柱侧弯的使用蜂窝介电谱的预后预测细胞为基础的检测协议

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

颈动脉和颈静脉导管在有意识大鼠模型进行血流动力学措施，输液和血液采样

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

在女小鼠害保留神经的中期尿道梗阻（NEMO）

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...