这项工作是由AO基金会（S-08-42G）和RISystem公司的支持。 我们想扩展一个非常大的"谢谢！"给Stephan Zeiter队在敖研究所达沃斯，瑞士正在让我们用自己的或设施为这个手术的拍摄如此宽松。

动物保健和实验方案如下，根据美国国立卫生研究院的指导方针，并经贝斯以色列女执事医疗中心实验动物管理和使用委员会，马萨诸塞州波士顿。 （协议编号:098-2009） 1，准备手术材料和仪器<ol><li>消毒用于执行使用前手术所有手术材料和仪器。包所需材料，使用或不使用工具托盘，折叠的布或纸包裹内有密封胶带釜蒸汽灭菌。将高压釜的温度应在125-135°C下的灭菌时间20-25分钟，干燥时间，然后10-15分钟。 </li><li>确保手术的时间只为200-250克。这是非常重要的，因为如果只在尺寸更重，那么不同尺寸的固定器应该被使用。对于只应使用重量超过250 GA较大的外固定器系统的版本。 </li> &lt;/ OL&gt; 2，手术过程和外固定器的应用<ol><li>任何动物的认证供应商购买只SD（或任何其他株）大鼠（男或女，200〜250克）。按照相应的动物护理和实验方案，根据已批准调查的机构动物照顾及使用委员会国家准则。允许在手术前至少48小时的适应期。 </li><li>对于手术，运送鼠到专用的手术房。 </li><li>麻醉与第一通过感应腔室的异氟醚的大鼠，然后继续在1-1.5升O 2 /分钟连接到麻醉机以1.5-2％的速度面罩。在手术开始时确保动物是在深度麻醉。要做到这一点，使用由延长肢体捏脚趾之间的网络用手指踏板反射技术（ 而不是脚趾本身！）。动物是不是苏如果肢体被撤回，肌肉抽搐发生，或者如果该动物使得噪声fficiently麻醉。 </li><li>大鼠是在深全身麻醉后进行手术，注射抗生素（头孢唑啉，20毫克/千克）和止痛的丁丙诺啡（剂量0.08毫克/千克）肌内右腿。避免了在手术过程中大量的流体损失，在之前的手术的身体重量3-5％的施用温暖的无菌盐水皮下，并且如果有必要在最后。应用无菌眼药膏眼睛，让他们水合，防止角膜损伤。 </li><li>下列药物注射剂，剃须和清洁用chlorohezadine或其他消毒溶液的大鼠的整个右后腿和动物转移到手术台。 （在腿部动作必须是相同的，将其用注射的人。） </li><li>将动物在俯卧位（ 图1A）加热表面。确保口罩留在鼻子和嘴向后ER转移到手术台，并保持在2.3中提到的麻醉方案）。用无菌窗孔被单悬垂的外科手术的区域，以便只腿用于手术被暴露。 </li><li>通过皮肤运行craniolateral从大转子到用手术刀（ 图1C）的膝关节的髁区域的右股骨的表面上作出的3-4厘米（ 图1B）的近似切口。通过轻柔解剖分离阔筋膜，并确保该肌肉组织不被切割露出股骨的轴。在这之后，分离分开的分枝股外侧肌和M.股二头肌和解除M.阔筋膜张肌，显露股骨的全长（在确认坐骨神经被保留; 图1D）。 </li><li>在截骨术的计划区域中，通过从FE释放周边的肌肉组织制备股骨沿骨干的中途区域MUR。首先，通过将Henahan电梯垂直于股骨的暴露表面，然后用手术刀时，释放在相邻区域中的肌肉启动。 <ol><li>通过向前推进继续到处去股骨，保持接近骨表面，直到所有的周边的肌肉组织从骨（如该缺陷将被创建）的整个中间部分被释放时，以及肌肉组织被完全清洗从骨。而这样做，这是非常重要的，以贴近骨表面，以避免切割任何主要血管。 </li></ol></li><li>对于一个5毫米大的骨缺损，环2个吉利线锯（0.22 MM）周围骨的内-外方向（ 图1E，F）。后循环的线锯，位置一块在股骨的远端侧，靠近膝关节，以及第二片上的基端侧靠近髋关节。夹紧吉利线锯，每边条采用S形弯曲的解剖和超级联赛茜镊子，使得它停留在所需的位置。如果一个切口截骨计划，那么只使用一块线锯。 </li><li>使用外固定器板作为模板，以确定所述植入物的精确位置。的外固定器的位置必须尽可能地靠近股骨的中心。 <ol><li>定位的外固定器板对骨的外侧表面。这是通过从外部旋转股骨实现。在该位置上的软组织层是在其最薄，从而防止过度的软组织张力固定器板下后的伤口闭合。 </li><li>然后稍稍抬起的外固定器板脱骨表面，以确保该板的孔中心向骨表面。持的外固定器具有小钳位为它留下平行于骨的纵向轴线，然后再使用电动工具或手钻预钻的第一孔上股骨与0.79的基端侧毫米的钻头。在向前发展，确保钻头的尖端仍集中于骨面。 </li><li>如果钻头的前端一直滑动，使用1.00毫米计数器沉降片（ 图8F），使第一个孔的位置。计数器沉降应用于定位所有剩余的安装销。这将确保在钻孔的相对于骨表面的精确对准和固定器板。 </li></ol></li></ol> 3，外固定支架植入方法采用SAW指南<ol><li>确保外固定器的板上锯导裁剪之前没有安装颠倒。通过比较在板中的孔的大小来确定。正确的一面是大孔的直径朝上。如果固定器内的孔的大小之间的差异并不明显，使用计数器沉降。 <ol><li>将反伸卡球的尖端插入孔上的一个固定器板，如果计数器沉降片容易装配到孔则是这种固定器的上侧，但是，如果计数器沉降片的前端不适合，那么这是固定器的底侧，并具有可翻转对于植入相应。 （ 重要提示:请确认钻垂直于骨的纵轴，因为这将确保固定器的完美定位于骨面的第一个钻孔的方向决定于骨骼的固定器的最终取向记住安装管脚具有相同的长度，并且如果固定器是不平行于骨的纵向轴线固定器与骨之间的距离会发生变化太多，可能会阻止所有四个安装管脚穿透两个皮层的能力。） </li><li>后的取向被证实，夹在板上的锯导（ 图2A，B），然后夹在装置上的骨，使得第一预钻孔对准在板（ 图2C）的第一孔。使用插入手电钻0.70平方毫米的套筒扳手带动第​​一安装销插入孔。这样做将允许可重复定位其余的安装引脚。 </li><li>后的第一个安装销是否到位，然后钻从在远端侧的第一安装销最远孔，并驱动所述第二安装销插入孔。的两个中间安装管脚注入顺序并不重要。 </li></ol></li></ol> 4，外固定支架植入方法没有锯指南: 也可以进行外固定器的应用程序，而无需使用该锯导。的外固定器植入的起始步骤是相同的​​，直到该单元与锯导被夹在骨头上（步骤3.1）。如果不使用该锯导，这是非常重要的，以保持该固定器板在正确的方向中整个应用程序。股骨需要在外侧方向外旋。 <ol><li>持的外固定器板用小夹子或S形弯曲的解剖和结扎镊子，使得它平行于骨（ 图3A）的纵向轴线。第一安装销的应用将确定固定器的对准，因此，骨的旋转必须被保留，直到第一销被插入时（ 图3B）。后的第一脚到位，小心使用镊子保持固定器板，以作为演练指导。 </li><li>插入钻头进入第二孔-这是最远侧孔的计划截骨间隙（ 图3C）。钻之前，检查并确保第二孔具有相同的方向作为第一个孔;还要确保后钻完成后，双方皮质参透。 </li><li>将0.70毫米见方的微博点¯x扳手的手钻，然后插入安装销插入小费。小心地将其插入到外固定器的板，而不会失去所述第一预钻孔的对齐。 </li><li>一旦该尖端与骨接触，开始转向下施加于手电钻的近端连续的轴向载荷的扳手。经过约5个圈，确保在固定销的近端线捕捉外固定架板的尸体。这个线程锁定系统。停止转动时，骨螺纹的端部是靠近骨（ 图3D）的顶表面上。 </li><li>后在最远端和近端侧上的插针的地方，预钻剩余的两个中间的孔。的中间的两个销植入顺序并不重要（ 图3C）。 </li><li>之后的外固定支架到位后，使用0.22毫米吉利线锯导演锯导，使节段性缺损（ 造型玩具ê4A）。如果选择后一种方法，该锯导板制造缺陷之前裁剪。 <ol><li>对于这一点，通过0.22mm的吉利线锯穿过股骨（ 图5A）下方的2槽使用充分灌溉（用5毫升注射器，免除盐水创造了往复运动一个5毫米的节段性缺损来回（ 图5B）缺陷创建的时间）。为了避免损坏软组织，切锯丝靠近骨的一侧完成截骨后。移除锯导板（ 图4B）。 </li></ol></li><li>缺陷或截骨被创建后，取出锯导和闭合伤口中的层时，肌肉第一（ 图4C），然后在皮肤（ 图4D）。在伤口闭合，治疗的缺陷按计划在研究方案。关闭肌层和使用Ethibond薇乔缝线4-0的阔筋膜，用爱惜康Monocryl 3-0 S中的皮肤uture。避免拖动缝合材料在非无菌表面，同时缝合伤口。注意:为了避免咬的伤口，缝合必须没有结束远端下植入。同样，可用于代替缝合皮肤胶。 </li><li>在前三术后几天，给大鼠镇痛每12小时以及抗生素每24个小时。当然，手术后的治疗方案的药物将取决于所使用的每个研究者的药物的制造商和品牌而有所不同（参照药物说明书中说明）。 </li><li>手术后经常监测动物，以确保他们从麻醉中恢复，然后才返回他们的住房设施。提供单独的住房，第几天手术后，确保没有任何并发​​症。 </li><li>手术后监测水，食物摄取和体重，以确保动物是不疼痛和痛苦。如果动物显示出活动水平降低，走动困难（可能植入失败），ataxiA，蓬头垢面苔白腻，眼睛周围和鼻孔，弯腰驼背的姿势，呼吸窘迫，食物和水的摄入减少等卟啉染色请教兽医。 </li></ol> 5，变更外固定支架刚度体内 <ol><li>若研究协议要求固定架刚度期间体内这个愈合过程的改变是通过改变固定使用附于手电钻0.5mm见方扳手盒的特殊互锁螺钉的连接元件来实现的。在此过程中，麻醉大鼠（参考在协议2.3），并给予镇痛（均指在协议2.4）只有一次的过程（ 图6A）的时间。 <ol><li>镇静的大鼠，然后插入0.50平方毫米的套筒扳手的前端进入安装于组装固定器侧的连动螺钉，并开始小心逆时针旋转，直到销是半出路（ 图6B </stron克&gt;）。之后这部分是完整的，则重复该过程为第二销即上的外固定器板（ 图6C）的同一侧。 </li><li>当两个引脚上的同一侧的一半出路，使用镊子或夹子来用温和的运动（ 图6D）除去在相对 ​​侧上的连接元件。该连接元件应该脱落容易，如果没有，则执行额外的夫妇匝上都互锁螺钉，以确保所述互锁螺钉的前端不会在连接元件埋入。 </li><li>该连接元件被移除之后，在地方的除去1（ 图6E）滑动所需的刚性连接元件，并从相反侧用方形框扳手开始旋转，直到互锁螺钉是从相反侧的一半（ 图6F）。重复同样的步骤进行第二联锁螺丝（ 图6G）。重要提示:次是将需要切换到该板的相对侧，以确保两个互锁螺钉是半出路在该处的连接元件被替换（图6H，I）的一侧。 </li><li>后此部分成功完成后，除去所述第二连接元件（ 图6J），它具有相同的刚度连接元件作为一个替代的相反侧（ 图6K）取代。后的第二连接元件是否到位，开始驱动互锁螺钉，直到联锁螺杆端离开该板的相对侧，并且所述互锁螺钉尖已经退出同量在每一侧（ 图6L）。重复同样的步骤进行第二联锁螺丝（ 图6M，N）。此过程需要约15分钟才能完成。 </li></ol></li></ol>

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B., 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=Direct+percutaneous+gene+delivery+to+enhance+healing+of+segmental+bone+defects.">Direct percutaneous gene delivery to enhance healing of segmental bone defects.</a> J Bone Joint Surg Am. 88, 355-365 (2006).</li><li>Cullinane, D. 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=Induction+of+a+neoarthrosis+by+precisely+controlled+motion+in+an+experimental+mid-femoral+defect.">Induction of a neoarthrosis by precisely controlled motion in an experimental mid-femoral defect.</a> J Orthop Res. 20, 579-586 (2002).</li><li>Dickson, G. 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笔者罗马诺马蒂斯是RISystem公司瑞士达沃斯产生的植入物，本文中使用的植入特定的仪器和耗材的雇员。笔者瓦伊达格拉特没有竞争的经济利益。

手术过程中最关键的步骤来创建一个大的骨缺损是:1）选择适当的体重的大鼠相匹配的外固定器的大小; 2）维持在手术过程中的无菌环境;和3）以下的外科手术过程的协议。 本研究的主要目标是设计，制造和对大鼠股骨大缺陷模型表征一个新的，可变刚性的外固定器，并使用此固定器在确定在愈合过程中的生物和机械因素之间的相互作用。新的定位器的机械性能进行了检查，?...

大量的研究已经提高了参与骨组织的修复1-6的生物学机制的理解。对骨修复的机械条件，如轴向，剪切和interfragmentary运动（的IFM）的影响已经被广泛地7-15研究。在过去的几年中，越来越多的研究开始出现用骨折，截骨术和大节段性骨缺损的体内模型，描述在骨愈合的力学环境的影响。因此，需要可靠的固定方法来获得可重复和可靠的研究成果。 周围的骨折愈合的力学环境是非常重要的，因为它决定的方式，将骨折愈合。因此，固定装置的选择是非常重要的，应根据不同的研究设计进行仔细选择，以及其它因素，如间隙大小和骨折的类型。该固定装置的机械性能再更重要的，当学习的大块骨缺损的骨性愈合，以建立一个固定的提供不仅遍及全重量的试验期带有一定的间隙大小，但也是一个理想的力学环境对骨愈合。外固定器在骨折和大骨缺损愈合的实验模型常用的，因为他们有比其他固定设备的优势。外固定器的主要优点是，它们允许在缺损部位在生物体内的机械环境的变化而二次干预，这可以通过在实验的过程中改变或调整该装置的稳定性杆来实现骨愈合的进展。此外，它允许对特定局部机械刺激的应用，以提高骨的修复，并且还提供了测量愈伤组织在体内的刚性的可能性。然而，这些设备也有一些缺点包括:软组织，感染和销断裂刺激。 不幸的是，这样的植入物是不提供"现成的"在植入物开发的时间，并调查被迫定制设计自己的固定器用于预期用途。因此，研究这方面的一个约束是缺乏实验控制中的大段缺损的局部力学环境，以及截骨术，因为它愈合。的外固定器的机械特性是由所定义，并且可以通过进行调制，大量的变量，包括:销，销的直径，销材料之间的距离，销，固定器杆的长度，固定器栏数的数目，固定架棒材，固定器杆的厚度和从骨表面到固定器杆（偏移）的距离。令人惊奇的是，研究仅缺乏可以发现，已经研究了各组分的机械贡献在啮齿类动物的研究16,18,28使用固定器或整体框架结构。例如，一项研究的结果表明，主要的因素，在确定的固定结构的总刚度之一是相对于它们的偏移量，直径和材料特性28把持销的灵活性。从上述研究结果清楚地表明，知道由固定装置提供的机械环境中是非常重要的，然而，在许多情况下，不详细调查。本文报道的设计，规格和植入体内的外固定器，解决这个问题的。此固定器还允许机械环境愈合的进展，一个属性，可以在体内治疗过程中的不同阶段的机械性刺激的敏感性的研究的调制。此外，还有强加控制的和可重复的当地技工人的环境中，其辅助功能也允许这样的环境中的骨愈合的不同阶段的调制。 我们设计的固定器是基于外固定，因而被广泛用于骨折固定16-21和大型缺损模型的实验动物22-27。我们的外固定架和文献报道的其他现有设计之间的不同之处在于它们的稳定性条用螺丝紧固有得紧用克氏针（克氏针）。这种类型的设计，需要螺钉被每两周重新拧紧（有时甚至每周一次），以确保该偏移的距离保持为加载是通过负重，以防止稳定性杆的松动施加。如果发生这样的松动，它允许不必要的额外负荷条件，如角，横向和扭转剪切动作，愈合骨（根据个人经验，与researche通信RS），认识到这一点，外部固定器被设计为使得需要改变固定器的刚度时，它会通过除去附着在那里的安装销被嵌入在主模块的连接元件来实现的。与新的外固定器样机进行体内试验试验，以确保其符合之前的大批量制造的所有提出的要求。 其主要目的为本文是提出一种新的手术方法，用于大的骨缺损和骨切开术的大鼠与在愈合过程中改变刚度体内的能力的外固定器。这种固定方法是在体内应用对大鼠股骨。

<table border="0" cellpadding="0" cellspacing="0" width="848">
	<tbody>
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			<td height="42" style="height:42px;width:232px;">Name of Material/ Equipment</td>
			<td style="width:283px;">Company</td>
			<td style="width:117px;">Catalog Number</td>
			<td style="width:216px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 100%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.120</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 70%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 40%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.121</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">RatExFix simple 10%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.122</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:232px;">RatExFix Connection element 100%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Connection element 70%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Connection element 40%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Connection element 10%</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.612.133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Main body</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.611.101</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix InterlockingScrew</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.412.110</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">RatExFix Mounting pin 0.85 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.412.100</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">RatExFix Saw Guide 100% 5 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.312.100</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Accu Pen 6V+</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.390.211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HandDrill</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.390.130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Drill Bit 0.79 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.593.203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Gigly wire saw 0.22 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.590.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Square box wrench 0.70 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.590.112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Square box wrench 0.50 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.590.111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Centering bit 1.00 mm</td>
			<td>RISystem AG Davos, Switzerland</td>
			<td style="width:117px;">RIS.592.205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Scalpel Blade handle</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Scalpel Blade (Size 15)</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tissue Forceps</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Scissors</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Retractor</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Needle Holder</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Henahan Elevator</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">S-shape curved dissecting and ligature forceps&#160;</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td>2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Dressing Forceps</td>
			<td style="width:283px;">Fine Science tools</td>
			<td style="width:117px;"></td>
			<td>2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sterile Fenestrated drape</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile gauze</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 ml syringe&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>&#160;for irrigation of defect</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-27G needle&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>&#160;for irrigation of defect</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1cc Insulin syringes&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for drug injections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile saline&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>for bone defect irrigation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sterile gloves</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>to perform surgeries</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chlorohezadine</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>disinfecting solution for surgical site</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vicryl suture 4-0 with SH-1</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>to suture muscle&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethibond suture 3-0&#160;</td>
			<td style="width:283px;">Fisher Scientific</td>
			<td style="width:117px;"></td>
			<td>to suture skin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isofluorine</td>
			<td style="width:283px;">Sigma-Aldrich</td>
			<td style="width:117px;"></td>
			<td>for anesthesia</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td style="width:283px;">Sigma-Aldrich</td>
			<td style="width:117px;"></td>
			<td>analgesia during and after the surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefazolin</td>
			<td style="width:283px;">Sigma-Aldrich</td>
			<td style="width:117px;"></td>
			<td>antibiotic during and after the surgery&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sprague-Dawley Rats or any other strain</td>
			<td>Charles River Laboratories International, Inc. (Wilmington, MA USA)&#160;</td>
			<td style="width:117px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

adjustable stiffness, external fixator for the rat femur osteotomy and segmental bone defect models

围绕断骨的愈合的力学环境是非常重要的，因为它决定的方式，将骨折愈合。在过去的十年里一直在通过病灶周围的固定稳定性改变力学环境改善骨愈合重要的临床利益。临床前动物研究在这方面的一个制约因素是缺乏实验控制了当地的力学环境中的大段缺损，以及他们截骨愈合的。在本文中，我们的设计和使用的外固定器的汇报，研究了大段骨缺损或截骨愈合。此设备不仅允许对骨病变可控制的轴向刚度，因为它可以治愈，但是它也使刚度在体内治疗过程中的变化的进行的实验表明，该固定器能够保持一个5毫米的股骨缺损间隙在不受限制的笼中老鼠体内活性为至少8周。同样，我们没有观察到变形或感染，包括在整个治疗期间销感染。这些结果表明，我们的新研制的外固定器能够实现可再现的和标准化的稳定化，并在大鼠体内大的骨缺损和各种尺寸截骨术的机械环境的改变。这证实了外固定器非常适合于使用在骨再生和修复的领域的大鼠模型的临床前研究调查。

设计规格大鼠股骨与外部固定系统的稳定化使得从0.5至5mm的创建截骨的。外固定支架系统是由聚醚醚酮（PEEK - [主体]）锁定外固定器和钛 - 铝 - 铌合金（TAN - [固定栓]），它提供了一个简单，重复性好，可调节的设计，并在四个不同的刚度可供选择:10，40，70和100％（100％为标准，最严格的固定器（ 图7）根据各研究者的研究需求，无论他们将不得不做植入刚度调?...

Watch this Scientific Journal Video about Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models at JoVE.com

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

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 ...

adjustable-stiffness-external-fixator-for-rat-femur-osteotomy

Research

JoVE Journal

Medicine

22.4K 次观看.  Queensland University of Technology. 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.

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

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

骨转移模型

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

科研

医学

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 ...

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

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

直接节段性肝内交付使用大鼠肝门部夹具模型方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

We present a robust protocol on how to carefully preserve and prepare cadaveric femora for fracture testing and quantitative computed tomography imaging. The method provides precise control over input conditions for the purpose of determining relationships between bone mineral density, fracture strength, and defining finite element model geometry and properties.

近端股骨尸制备基于CT的断裂强度测试和定量的有限元分析

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength.  Because ex vivo biological tissues are prone to ...

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 ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

用膝关节关节仪评价大鼠膝关节屈曲收缩的特定组织贡献

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

大鼠胡萝卜动脉压力控制段气球损伤与腹腔ventit 治疗应用

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.

Here, we present a protocol to iatrogenically fracture the shaft of the femur of Wistar albino rats and follow up on the development of the callus. This femur osteotomy model can help researchers evaluate the process of fracture healing and to study how a drug could influence fracture healing.

开发Wistar白化大鼠股骨截骨模型的协议

Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. ...

Methods Article

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