Name: an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice
Uploaded: 2016-11-13T15:00:00.000+00:00
Duration: 7 min 41 s
Description: Watch this Scientific Journal Video about An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice at JoVE.com

这项工作是由RISystem AG，达沃斯，瑞士的支持。

所有的程序都是IACUC认可和遵循机构准则（Landesamt献给Verbraucherschutz，ZentralstelleAmtstierärztlicher丁斯特，萨尔布吕肯，德国）。镇痛和感染预防应与那里的实验是要执行的国家和机构的相应准则协议。 1.植入物和手术器械的研制<ol><li>选择手术刀刀片（尺寸15），小准备剪刀，细镊子，敷料镊子，小镊子，24号（G）和27克针状物，一种非可再吸收的5-0缝线，和一个针保持器从显微仪器框。 </li><li>解压髓内钉，联锁销，特别瞄准装置中，吉利看到，对于吉利模板看到的，定心钻头（直径为1mm），钻头（直径0.3mm），以及手钻（ 图2;见材料清单）。 注:intramedullary钉（0.8毫米直径15.7毫米的长度）为逆行植入股骨由医用级不锈钢的髓内锁钉。钉具有近端螺纹（4毫米长度）和联锁销的插入（0.3毫米直径），以实现轴向和旋转稳定性的两个孔（ 图1）。 </li><li>露出植入物和所有外科器械的消毒溶液（ 例如，96％醇）5分钟或消毒它们（蒸汽灭菌，130℃，25分钟）。消毒或灭菌后，将仪器在无菌操作下布。直接相邻的小动物动作表的无菌操作布的位置。 </li></ol> 2.动物，麻醉，镇痛和<ol><li>选择菌株，年龄，并在必要时为研究和解决的问题的小鼠的性别。 备注:本研究12至14周龄雄性CD-1小鼠。指甲implantation，理想体重的动物是25 - 35克</li><li>麻醉小鼠以15毫克/千克甲苯噻嗪和75毫克/千克氯胺酮腹膜内注射。确认通过脚趾捏的麻醉。敷眼润滑剂保护动物的眼睛从麻醉中干燥。麻醉诱导后，请将鼠标热灯下以保持体温恒定。 </li><li>在手术之前，手术后第3日之前申请中的饮用水（2.5毫克/ 100毫升），用于从第1天镇痛曲马多盐酸盐。 </li></ol> 3.手术程序和指甲植入<ol><li>手术前，剃了整个右后腿和应用脱毛膏。 5分钟后，取出奶油和用水清洗的腿。露出植入物和所有外科器械的消毒溶液（ 例如 ，96％的醇）或消毒它们（蒸汽灭菌，130℃，25分钟）。 </li><li>在无菌条件下，将备忘录硒对小动物手术台仰卧位。弯曲右膝关节，以允许前路到膝盖的髁。在使用手术刀刀片右膝执行一个5毫米的内侧髌切口。 </li><li>提起与细镊子的髌韧带，并与手术刀刀片小心地调动了韧带。然后，用手术刀刀片横向移动髌骨以暴露股骨的髁间凹口。 </li><li>通过钻，直至到达骨髓腔内打开髁间凹口。 <ol><li>开始一个45º偏移使用1mm的定心钻头股骨轴钻。钻井，直到它平行于股骨的骨轴期间慢慢地改变钻头的方向。停止，如果到达骨髓腔钻井。 </li></ol></li><li>在间切迹打开骨后，插入24克针插入在股骨的整个长度的髓内腔中。铰的intrame股骨dullary腔通过24克针的旋转运动手动。取出24克针插入较薄27克针进入骨髓腔。推针前进到穿孔股骨的皮质骨近端的大转子。 </li><li>取下股骨27克针。使用手电钻，通过下连续旋转和轴向压力间切迹植入髓内钉，直到钉的远端到达髁的水平。 注:该钉的远端可以具有小标记来识别。 </li><li>放置在左侧卧位鼠标。为了手术暴露股骨的中段执行使用沿从膝关节的髋关节的侧向股骨的骨干部分的手术刀刀片的纵向皮肤切口。 </li><li>使用小准备剪刀，分裂筋膜和蔓延的肌肉在从侧方的股骨轴的方向。传播的肌肉直到股骨的骨干部分露出。保留坐骨神经。 <ol><li>通过破坏与敷料镊子骨准备股骨的整个圆周。然后，通过传播敷料镊子收缩的肌肉和揭露股骨。 </li></ol></li><li>挂载瞄准装置到骨钉的远端。推进装置，直到它连接到骨钉的适配器凸缘，并打开瞄准装置中外侧位置到股骨。 </li><li>互锁带近端和远端的联锁销钉。 <ol><li>开始与近端连动销。 </li><li>将定心钻头（1毫米直径）插入手钻。埋头在近端互锁孔位的骨头。 注:锪孔，一个小腔体在面临骨皮质创建不通过骨钻孔。该空腔允许用于改善定心和（直径0.3毫米）引导所述稀释剂钻头，以后使用。 </li><li>插入钻头（0.3毫米直径）插入手钻。使用瞄准装置，通过表面和避免皮质骨（双皮质）两个钻孔。通过瞄准装置插入第一连动销。联锁销驱动轴剪断，一旦互锁扭矩来实现的。 </li><li>重复此过程为远端连动销。 </li></ol></li><li>执行骨干截骨术。 <ol><li>附加锯导向瞄准装置上的两个互锁销之间的横向侧。然后，看到了骨与吉利在连续灌溉用生理盐水看到。截骨完成后，切锯在一端，靠近骨。小心地取出锯，以避免造成对软组织损伤。 </li></ol></li><li>除去瞄准装置，并与小钳子，夹断髓内钉的剩余轴在标记行。 </li><li>关闭肌肉层在北纬股骨全部擦除网站并进行皮肤缝合单缝线。在膝盖的前部位，重新定位髌骨并固定髌骨腱与一个单一缝合肌肉。使用单缝合关闭这个伤口为好。 </li><li>保持温暖的灯光下的动物，直到他们从麻醉中恢复。不要让动物无人看管，直到他们重新获得足够的意识，保持腹卧。返回动物单个笼中的动物设施。 </li><li>每天仔细监测动物。期间的前三天保持术后镇痛。继续镇痛如果在手术后第4天，动物仍然显示疼痛的迹象，通过发声，烦躁，缺乏流动性，没有新郎，异常姿势和缺乏正常的环境感兴趣的指示。终止镇痛当动物无痛苦。 </li></ol>

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Romano Matthys is an employee of RISystem AG, Davos, Switzerland, which produces the implants and implant-specific instruments. The other authors have no conflicts of interest.

手术技术的最重要的步骤是钉，瞄准装置，和销的正确定位。钉已被完全插入到在骨钉的远端标记缩进，因为钉的突出成在髁的水平的膝关节可以限制膝关节（ 图3A）的移动。因此，股骨的大小，因此，动物的体重，必须加以考虑。外科医生还应该特别注意的瞄准装置安装钉的连接法兰的最终位置。这保证了截骨总是在一个相同的中段位置。最后，联锁销必须被完全bicortically插入，以避免在观察期间（ 图3 B）的销错位。因此，销应该插入使用旋转运动和连续的轴向载荷的股骨。引脚传动轴剪关只要互锁扭矩来实现的。钉子和针的最终位置必须通过X线进行确认的动物被纳入研究协议之前。 在实验结束时，骨收获期间的最关键的步骤是去除从指甲联锁销。相当频繁，销的端部是由新形成的骨组织覆盖。事实上，这种骨组织已被切除，直到销可被除去。这必须非常小心地进行，因为到股骨任何损坏能够影响愈合骨的生物力学特性。有时，销可以更容易地从股骨的背侧去除。指甲本身可以不使用简单的缝合针夹持器的任何困难地除去。 此手术过程的器械和植入物是高度特异性的，所以修改的程序不能进行。虽然该过程可能会开发COM褶皱，在我们的手中，他们是罕见的（ 即低于2％）。例如，髌韧带，这是在手术过程中横向移动时，可能会破裂。这需要钉植入后韧带缝合。在24克针和股骨的扩孔的插入，髁爆裂。在针插入和截骨与吉利所看到的，股骨可在中段区域的突破。没有解决问题，这些并发症的可能性，因此，这些动物不能用于标准化实验。 该技术的一个限制是，动物的大小不同，因此，股骨，需要植入物的不同的尺寸。关于使用所述植入物的另一个限制是， 在体内显微CT在愈合过程中几乎是不可能的截骨分析由于植入物材料（医用级不锈钢），这将影响图像质量。 体内骨愈合的研究可以使用开放或封闭的模式下进行。在大多数研究中，股骨的愈合或胫骨进行了分析。这适用于在过去十年中小鼠开发的技术和模式真。这些措施包括开放7-10模型以及封闭5,11,12的方法，它可以提供刚性或者较小刚性的固定。有趣的是，在小鼠以前的研究用一个简单的髓内钉。虽然这样一个简单的引脚与巨大骨痂形成有关稳定骨折愈合，技术承担了相当数量的缺点。这些措施包括针错位由于轴向和旋转稳定性故障异构愈合反应。虽然这些缺点是已知的影响的实验结果，最近的研究，其打算分析骨愈合的机制，仍使用小鼠模型，其中所述断裂是用销13仅稳定或甚至留下未稳定<s达&gt; 14。因此，由于显着的愈合过程中力学影响愈合15的过程中，我们觉得稳定接骨技术，可比那些在临床实践中使用，也应该用于小鼠。 相比于简单的髓内钉的当髓内锁钉的费用是相当高的。然而，销承载错位的危险，并且不提供轴向和旋转稳定性。这可能会影响结果的质量，并需要的动物用于研究的更大数量。与此相反，髓内锁钉允许截骨术和骨缺损的稳定具有高度标准化的，导致的结果降低可变性。这导致在动物的必要数量的减少。 克服了常用销的轴向和旋转的不稳定，几个植入物已在过去的几年中引入的。这些包括髓内钉，它通过一个特别改性远端头和近侧线5诱导断裂的压缩。 必要植入螺钉的外科技术是简单且比该髓内钉的侵入性更小。然而，螺杆示出相比于钉4旋转刚度更低。此外，它不能因轴向稳定性是通过横跨骨折的骨碎片的轴向压缩来实现用作缺陷愈合的模型。 内部锁定板，其可用于刚性固定，导致骨愈合，这是由膜内骨化9为主。因为这种类型的愈合用愈伤组织形成小相关联，该模型可能无法在需要用于生化和分子分析更大量的愈伤组织的实验优选的。的兴趣，内部锁定板也可以更换esigned对更灵活的固定技术16。使用这种挠性板，骨愈合是由软骨内骨化为主，从而导致更大量的愈伤组织。然而，骨痂形成是异类，在工地对面的板布局主要发生。锁定板也允许骨缺损的稳定化。然而，间隙的大小是有限的，不会导致一个可靠的非联合形成6。 小鼠的外固定器，提供用于分析骨缺损愈合良好定义的替代钉。相比，这里介绍了钉的外固定器的主要优点是，以验证骨愈合17期间在体内植入物的刚性的可能性。然而，销的感染和正常的身体活动的改变，由于外部施加的固定部件也必须加以考虑。 特别感兴趣的，既不是整型ernal锁定板也没有的外固定器允许不同的骨替代物和组织工程构建体，其可以在骨缺损愈合待分析的标准化固定。当使用这两种技术，骨替代品或组织工程构建体必须被放置到缺陷，这通常需要额外的固定18。与此相反，使用了钉允许骨替代在指甲的注入，提供充分的定位和固定（ 图6）。 这里介绍的髓内锁定钉比得上用于在临床实践中治疗创伤的患者的指甲。因此，我们认为，可以钉在小鼠骨愈合的研究范围从正常到有缺陷的广泛使用。

骨愈合的生物学可在体外使用细胞和球体培养物进行研究，但它也需要在体内使用动物研究的方法。而大的动物实验还处于临床前试验中发挥重要作用，产品或假设早期测试已经在过去的10年变化，并且现在通常在小动物模型1进行。有几个原因，进行这一切换。生产和小鼠和大鼠的维护都比较便宜猪和羊。此外，小动物具有较短再现时间和更短的正常愈合期间，这两种促进大系列慢性实验的性能。最后，基因靶向的动物和特异性抗体的可用性允许在骨愈合的分子机制的分析。然而，虽然预先在较大的动物模型用于骨接合术的技术可以用最小variat翻译<html>从人或兽医临床病人的护理，在小尺寸的大鼠和小鼠的发展和骨接合术的技术应用中使用类似的程序离子竟然是具有挑战性的。 这是众所周知的，该生物力学环境显著影响骨愈合过程2。从骨折愈合在人类已知的，在治疗的不同的模式，包括与micromovements较小刚性固定后固定牢靠和软骨内骨化后膜内成骨骨折的稳定结果的差异。完整的轴向或旋转不稳定可能延迟愈合过程或可能导致非工会3。因此，我们认为，有必要制定在小鼠和大鼠复杂植入系统和接骨技术。以这种方式，生物力学条件可以适当地标准化，以保证有效的结果分析愈合过程时。 e_content"&gt;虽然有相当数量的高度复杂的鼠稳定技术已在过去的几年中已经引入，最常用的技术仍是简单的髓内钉。这个技术的主要缺点，但是，是缺乏的旋转和轴向稳定性4，为了提高旋转和轴向稳定性，髓内螺钉被引入，以稳定在小鼠中5股骨骨折。然而，螺钉固定，不能用来分析骨缺陷的愈合由于需要在接触和压缩所述骨碎片之间为了保持旋转稳定性。 髓内锁钉提供更高的轴向和旋转稳定相比简单的引脚和髓内螺钉4。高度重复性的股骨截骨术，可能的，因为指南的吉利锯，并创造界定的间隙大小的能力，允许正常苯教的分析Ë愈合和骨愈合不良6。由于联锁销的插入，髓内锁钉保证在整个治疗过程中的恒定间隙的大小，即使在轴承全部重量。这里，我们的设计和髓内锁定钉的应用报告，以及它的优点，以及对正常和延迟骨愈合实验研究的缺点。

<table><tbody><tr><td>MouseNail</td><td>RISystem AG</td><td>221,122</td><td></td></tr><tr><td>MouseNail aiming device</td><td>RISystem AG</td><td>221,201</td><td></td></tr><tr><td>MouseNail interlocking pin</td><td>RISystem AG</td><td>221,121</td><td></td></tr><tr><td>Centering bit</td><td>RISystem AG</td><td>592,205</td><td></td></tr><tr><td>Drill bit</td><td>RISystem AG</td><td>590,200</td><td></td></tr><tr><td>Gigli wire saw</td><td>RISystem AG</td><td>590,100</td><td></td></tr><tr><td>Suture (5-0 Prolene)</td><td>Ethicon</td><td>8614H</td><td></td></tr><tr><td>Forceps</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BD520R</td><td></td></tr><tr><td>Dressing forceps</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BJ009R</td><td></td></tr><tr><td>Scissors</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BC100R</td><td></td></tr><tr><td>Needle holder</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>BM024R</td><td></td></tr><tr><td>24 G needle</td><td>BD Mircolance 3</td><td>304100</td><td></td></tr><tr><td>27 G needle</td><td>Braun Melsungen AG</td><td>9186182</td><td></td></tr><tr><td>Scalpel blade size 15</td><td>Braun Aesculap AG &amp;amp;CoKG&amp;nbsp;</td><td>16600525</td><td></td></tr><tr><td>Pincers</td><td>Knipex</td><td>7932125</td><td></td></tr><tr><td>Heat radiator</td><td>Sanitas</td><td>605.25</td><td></td></tr><tr><td>Depilatory cream</td><td>Asid bonz GmbH</td><td>NDXZ10</td><td></td></tr><tr><td>Eye lubricant</td><td>Bayer Vital GmbH</td><td>2182442</td><td></td></tr><tr><td>Xylazine</td><td>Bayer Vital GmbH</td><td>1320422</td><td></td></tr><tr><td>Ketamine</td><td>Serumwerke Bernburg</td><td>7005294</td><td></td></tr><tr><td>Tramadol</td><td>Gr&amp;uuml;nenthal GmbH</td><td>2256241</td><td></td></tr><tr><td>Disinfection solution (SoftaseptN)</td><td>Braun Melsungen AG</td><td>8505018</td><td></td></tr><tr><td>CD-1 mice</td><td>Charles River</td><td>22</td><td></td></tr></tbody></table>

an intramedullary locking nail for standardized fixation of femur osteotomies to analyze normal and defective bone healing in mice

骨愈合模型是新的治疗策略的发展为临床骨折的治疗是必不可少的。此外，小鼠模型正在成为创伤研究更常用。它们提供了大量的突变株和抗体对骨愈合的高度分化的过程背后的分子机制的分析。为了控制生物力学环境，规范和良好的特点接骨技术在小鼠强制性的。在这里，我们的设计和使用的髓内钉的报告在小鼠体内稳定开放股骨截骨。钉，由医用级不锈钢的，提供高的轴向和旋转刚度。所述植入物进一步允许的限定，恒定截骨间隙创建尺寸从0.00毫米到2.00毫米。髓内锁定股骨截骨钉镇定的0.00和0.25mm间隙尺寸导致通过软骨内和膜内ossificat足够骨愈合离子。与萎缩性不愈合2.00毫米结果的间隙大小股骨截骨的镇定。因此，髓内锁钉可在愈合和未愈合的机型。使用相比其他开放骨愈合模型指甲的另一优点是，以充分修正骨代用品和支架为了研究骨整合的过程中的可能性。使用该髓内钉的缺点是愈创手术过程，固有相比封闭模型所有打开的程序。另一个缺点可能是骨髓腔一定的伤害的诱导，固有相比髓外稳定程序的所有髓内稳定技术。

用于外科手术的总时间为从皮肤切口约30分钟至伤口闭合。使用所提供的外科植入物，可在没有立体显微镜来进行手术。术后，动物每天监测。术后镇痛3天后终止，因为没有动物表现出这一段时间后疼痛（发声，烦躁，缺乏流动性，不培训，不正常的姿势，或缺乏周围正常利息的）证据。动物手术后显示出2天内正常负重。在整个观察期内未观察到伤口感染或继发性骨折。 可能发生的最重要的并发症是锁紧钉的不正确的植入，用指甲水平与膝关节（ 图3 A）的髁的突起。这主要发生原因可能会瞄准装置的不正确的操作或由于使用的动物用太小的股骨，特别是在与体重低于20克小鼠。另一个复杂的互锁销（ 图3 B）中的位错。这种并发症可以通过正确的种植体植入放射确认过程中或手术后立即被避免。这个问题主要是由于针的不完全插入。最后，骨收获在实验结束时阻碍了几次，因为它是难以除去联锁销。这是由于周围的骨针位置桥接。 放射分析5周后确认的0.25毫米截骨差距完全愈合。在这个时间点，骨膜愈伤组织几乎完全改造（ 图4的A）。相反，在一个2.00毫米差距稳定股骨，截骨不愈合。股骨可靠地显示出<html> ñ萎缩性不愈合形成。这10周的骨愈合（ 图4 B）后也证实。 稳定与0.25mm的间隙截骨后，组织学分析表明继发性骨折愈合的骨痂形成，包括膜内和软骨内骨化一个典型的模式。后5周，截骨完全被骨组织桥接。在这个时间点，编织骨已经被改造成层状骨（ 图5的A）。与此相反，股骨稳定与2.00毫米截骨间隙10周后观察表明萎缩性不愈合。这是与截骨间隙内高量的纤维组织相关联。在截骨术中没有显示骨愈合或组织学时分析弥合的迹象（ 图5 B）。 2 / 54472fig1.jpg"/&gt; 图1:植入物。答髓内钉（直径0.8mm 15.7 mm长）与近端螺纹（箭头4毫米的长度）和两个孔（箭头头部），用于联锁销的插入。钉连接到一个轴（双箭头），以促进应用。B.联锁销（直径0.3mm，箭头），以实现旋转和轴向稳定性。该连动销也连接到一轴（双箭头），方便的应用程序。C.髓内钉植入到小鼠股骨后， <a href="http://ecsource.jove.com/files/ftp_upload/54472/54472fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54472/54472fig2.jpg" /> 图 2: 钉植入手术器械。 A.针对设备用于插入指甲。B.锯导要用于创建截骨为0.25毫米的间隙尺寸。C.钻头钻的孔的联锁销。D.定心钻头的联锁销孔的锪用于指甲，锪孔，钻孔，及联锁销插入的插入。E.手电钻， <a href="http://ecsource.jove.com/files/ftp_upload/54472/54472fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54472/54472fig3.jpg" /> 图3: 术后X光片。答射线照相展示钉的突出部（箭头）到膝关节的髁的水平。B.射线照相展示近端interloc的不完全插入主销（箭头）。比例尺代表4毫米。 <img alt="图4" src="/files/ftp_upload/54472/54472fig4.jpg" /> 图4:5 10周的骨愈合后射线照片。用0.25mm的间隙截骨稳定股骨A. X线分析，后5周，显示出足够的骨愈合。有2.00毫米截骨间隙稳定10周后股骨B. X线分析，表明萎缩性不愈合。比例尺代表4毫米。 <img alt="图5" src="/files/ftp_upload/54472/54472fig5.jpg" /> 图5:5 10周的骨愈合后组织切片。用0.25mm的截骨间隙稳定后5周股骨的A.组织学分析，证明充分的骨愈合。注意用板层骨几乎完全的重塑。B.组织胺用2.00毫米截骨间隙稳定10周后股骨的ological分析，表明萎缩性不愈合。注意在截骨间隙的纤维组织。组织学切片，根据所述三色法染色。比例尺代表800微米。 <img alt="图6" src="/files/ftp_upload/54472/54472fig6.jpg" /> 图6:骨替代植入 体内的照片展示了鼠标的右股骨一个段骨缺损。缺陷是由骨替代（箭头）填补。骨替代植入过指甲，提供足够的定位和固定。

Watch this Scientific Journal Video about An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice at JoVE.com

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

髓内锁钉股骨截骨标准化固定用于分析正常和有缺陷的骨愈合小鼠

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

an-intramedullary-locking-nail-for-standardized-fixation-femur

Nanyang Technological University

Research

JoVE Journal

Medicine

13.3K 次观看.  Saarland University. This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

视频: An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification, Distraction Osteogenesis, or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens4. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice 5-7, after application of bioactive molecules 8,9, after altering physiological parameters of healing 10, after modifying the amount or time of stabilization 11, after distraction osteogenesis 12, after creation of a non-union 13, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.

This article describes a method for stabilizing long bone fractures that is based on the application of modified Ilizarov external fixators 1-3. After application of the fixators and creation of the bone injury, healing can be assessed, distraction osteogenesis can be performed, or non-union or critical sized defect can be created and used to study therapeutic interventions.

建立评估膜内骨化硬性稳定骨折，牵张成骨，或临界中小企业缺损愈合

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role ...

科研

医学

A Mini-Invasive Internal Fixation Technique for Studying Immobilization-Induced Knee Flexion Contracture in Rats

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to restrict knee joint mobility is a widely accepted model to generate experimental contracture. However, implanting application will inevitably cause surgical trauma to the animals. Aiming to develop a less invasive approach, we combined a muscle-gap separation modus with a previously reported mini-incision skill during the surgical procedure: Two mini skin incisions were made on the lateral thigh and leg, followed by performing muscle-gap separation to expose the bone surface. The rat knee joint was gradually immobilized by a preconstructed internal fixation at approximately 135&#176; knee flexion without interfering essential nerves or blood vessels. As expected, this simple technique permits rapid postoperative rehabilitation in animals. The correct position of the internal fixation was confirmed by an x-ray or micro-CT scanning analysis. The range of motion was significantly restricted in the immobilized knee joint than that observed in the contralateral knee joint demonstrating the effectiveness of this model. Besides, histological analysis revealed the development of fibrous deposition and adhesion in the posterior-superior knee joint capsule over time. Thus, this mini-invasive model may be suitable for mimicking the development of immobilized knee joint contracture.

Here, we present a protocol to describe a minimally invasive technique for knee joint immobilization in a rat model. This reproducible protocol, basing on muscle-gap separation modus and the mini-incision skill, is suitable for studying the underlying molecular mechanism of acquired joint contracture.

研究大鼠固定性诱导膝关节弯曲收缩的微型侵入性内固定技术

Joint contracture, resulting from a prolonged joint immobilization, is a common complication in orthopedics. Currently, utilizing an internal fixation to ...

发育生物学

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 ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

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 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 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 ...

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

The reliable generation of consistent stabilized fractures in animal models is essential for understanding the biology of bone regeneration and developing therapeutics and devices. However, available injury models are plagued by inconsistency resulting in wasted animals and resources and imperfect data. To address this problem of fracture heterogeneity, the purpose of the method described herein is to optimize fracture generation parameters specific to each animal and yield a consistent fracture location and pattern. This protocol accounts for variations in bone size and morphology that may exist between mouse strains and can be adapted to generate consistent fractures in other species, such as rat. Additionally, a cost-effective, adjustable fracture apparatus is described. Compared to current stabilized fracture techniques, the optimization protocol and new fracture apparatus demonstrate increased consistency in stabilized fracture patterns and locations. Using optimized parameters specific to the sample type, the described protocol increases the precision of induced traumas, minimizing the fracture heterogeneity typically observed in closed-fracture generation procedures.

The goal of the protocol is to optimize the fracture generation parameters to yield consistent fractures. This protocol accounts for the variations in bone size and morphology that may exist between animals. Additionally, a cost-effective, adjustable fracture apparatus is described.

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 ...

Transverse Fracture of the Mouse Femur with Stabilizing Pin

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 whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

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 ...