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 border="0" cellpadding="0" cellspacing="0" width="476">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:187px;">MouseNail</td>
			<td style="width:179px;">RISystem AG</td>
			<td style="width:111px;">221,122</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail aiming device</td>
			<td>RISystem AG</td>
			<td>221,201</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MouseNail interlocking pin</td>
			<td>RISystem AG</td>
			<td>221,121</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Centering bit</td>
			<td>RISystem AG</td>
			<td>592,205</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Drill bit</td>
			<td>RISystem AG</td>
			<td>590,200</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Gigli wire saw</td>
			<td>RISystem AG</td>
			<td>590,100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture (5-0 Prolene)</td>
			<td>Ethicon</td>
			<td>8614H</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BD520R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dressing forceps</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BJ009R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BC100R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle holder</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>BM024R</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">24G needle</td>
			<td>BD Mircolance 3</td>
			<td>304100</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">27G needle</td>
			<td>Braun Melsungen AG</td>
			<td>9186182</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scalpel blade size 15</td>
			<td>Braun Aesculap AG &#38;CoKG&#160;</td>
			<td>16600525</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pincers</td>
			<td>Knipex</td>
			<td>7932125</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Heat radiator</td>
			<td>Sanitas</td>
			<td>605.25</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Depilatory cream</td>
			<td>Asid bonz GmbH</td>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; NDXZ10</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Eye lubricant</td>
			<td>Bayer Vital GmbH</td>
			<td>2182442</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazine</td>
			<td>Bayer Vital GmbH</td>
			<td align="right">1320422</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ketamine</td>
			<td>Serumwerke Bernburg</td>
			<td align="right">7005294</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tramadol</td>
			<td>Gr&#252;nenthal GmbH</td>
			<td align="right">2256241</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Disinfection solution (SoftaseptN)</td>
			<td>Braun Melsungen AG</td>
			<td align="right">8505018</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">CD-1 mice</td>
			<td>Charles River</td>
			<td align="right">22</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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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="https://ecsource-jove-com.remotexs.ntu.edu.sg/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

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

采用实时定量PCR检测供体细胞植入一个具有竞争力的小鼠骨髓移植模型

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

科研

医学

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

病变资源管理器:一个视频引导，标准化为老年痴呆症准确，可靠的MRI衍生Volumetrics和正常老年人协议

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

可调节刚度，外固定支架的大鼠股骨截骨和节段性骨缺损模型

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

股骨骨髓的愿望在活体小鼠

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the silicone compound, via a 26 G catheter, into the portal or hepatic vein. After silicone injection, organs were explanted and prepared for ex-vivo micro-CT (&#181;CT) scanning. The silicone injection procedure is technically challenging. Achieving a successful outcome requires extensive microsurgical experience from the surgeon. One of the challenges of this procedure involves determining the adequate perfusion rate for the silicone compound. The perfusion rate for the silicone compound needs to be defined based on the hemodynamic of the vascular system of interest. Inappropriate perfusion rate can lead to an incomplete perfusion, artificial dilation and rupturing of vascular trees.
The 3D reconstruction of the vascular system was based on CT scans and was achieved using preclinical software such as HepaVision. The quality of the reconstructed vascular tree was directly related to the quality of silicone perfusion. Subsequently computed vascular parameters indicative of vascular growth, such as total vascular volume, were calculated based on the vascular reconstructions. Contrasting the vascular tree with silicone allowed for subsequent histological work-up of the specimen after &#181;CT scanning. The specimen can be subjected to serial sectioning, histological analysis and whole slide scanning, and thereafter to 3D reconstruction of the vascular trees based on histological images. This is the prerequisite for the detection of molecular events and their distribution with respect to the vascular tree. This modified silicone injection procedure can also be used to visualize and reconstruct the vascular systems of other organs. This technique has the potential to be extensively applied to studies concerning vascular anatomy and growth in various animal and disease models.

Tools used for visualizing vascular regeneration require methods for contrasting the vascular trees. This film demonstrated a delicate injection technique used to achieve optimal contrasting of the vascular trees and illustrate the potential benefits resulting from a detailed analysis of the resulting specimen using &#181;CT and histological serial sections.

之后，正常小鼠70％部分肝切除血管和实质再生的可视化

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

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 ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...