作者承认美国国家卫生研究院（NIH）国家关节炎和肌肉骨骼和皮肤病国家研究所的资助，授权号为R03AR068087，科罗拉多大学医学院学术浓缩基金和盖茨再生医学中心。这项工作也得到NIH / NCATS科罗拉多CTSA授权号UL1 TR001082的支持。内容是作者的唯一责任，不一定代表NIH的正式观点。

所有动物手术必须得到当地机构动物保护和使用委员会（IACUC）的批准。以下程序的动物方案由科罗拉多大学丹佛IACUC批准。 获得大鼠注意:除非需要转基因动物，否则在手术时需要6周龄的骨骼不成熟的Sprague-Dawley大鼠。可能使用其他菌株;然而，大多数已发表的研究已经在Sprague-Dawley大鼠上进行。 2.手术用品的准备<ol><li>高压灭菌手术供应包包括以下各项:＃3手术刀手柄，针架，Adson镊子和虹膜剪刀。 </li><li>高压灭菌无钥匙钻卡盘。当在多只动物上操作时，钻夹头可能会在动物手术之间进行珠粒灭菌。 注意:关于使用无菌手术的本地IACUC规则必须遵守多种动物的手段。例如，科罗拉多大学丹佛IACUC允许一种外科手术套件在停用前最多可以使用5只动物。此外，手术工具必须使用动物之间的珠粒灭菌器进行热灭菌。额外的无菌手术包必须用于任何额外的动物。 </li><li>高压灭菌5厘米Steinmann针，每只动物一只。 注意:为了降低感染的风险，Steinmann针不能用于多只动物。 </li><li>高压灭菌器1.8毫米牙科钻头，每个动物一个。 注意:为了减少感染的风险，牙科医生不能用于多种动物。 </li><li>如果适用，高压灭菌夹施加器。或者，可以使用埋置的缝合线封闭皮肤层。请参阅步骤7.3。 </li><li>如果可能，使用辐射或气体灭菌消毒旋转钻。 </li><li>收集以下附加用品:电动剃须刀无菌纱布，聚维酮碘，无菌盐水，无菌10ml注射器，无菌23号针，异丙醇拭子，异氟烷，卡尺，手术后镇痛药（ 如 NSAIDs和丁丙诺啡），无菌外科手术，无菌手术手套，无菌＃15手术刀刀片，无菌伤口夹，麻醉机，珠粒灭菌器，加温垫和吸收垫。 </li></ol>麻醉和动物的制备<ol><li>通过将动物引入1到2L的诱导室，通过带有被动清除系统的汽化系统，从1升/分钟的氧气流中加入5％异氟烷，将动物麻醉。 注意:接触5％异氟烷应在5分钟内麻醉6周龄的老鼠。 </li><li>将动物移动到手术部位，并使用鼻锥在2〜3％的异氟烷​​麻醉下保持动物的余下时间。将动物仰卧在暖气垫和吸收物上进入下垫。 注意:动物不需要固定在手术台上。按照下面的步骤握住腿是足够的稳定方法。 注意:所有后续程序将在动物麻醉下进行。 2-3％异氟烷应足以维持在这个年龄的大鼠麻醉。这可以通过测试双足戒断反射来证实。 </li><li>根据制度上认可的政策（ 例如 ，0.05 mg / kg的丁丙诺啡和5 mg / kg的卡洛芬）来管理术中止痛药。 </li></ol> 4.准备胫骨外科手术<ol><li>用电动剃须刀将整个后腿从内侧的踝部刮到骨盆。 </li><li>使用卡尺测量并记录胫骨前部的胫骨长度到内踝的下侧。或者，使用X射线或微CT 11测量整个胫骨长度</<html> sup&gt; ， 12，14 。任选地，使用X射线或微CT在手术前测量生长板尺寸。 </li><li>用酒精拭子擦拭整个腿部，腹部和生殖器，然后用聚维酮碘浸泡的纱布清洁手术部位。 注意:为了最大限度地减少感染的风险，必须在无菌条件下，直到动物从麻醉中去除（步骤7.4）为止。所有外科手术材料都必须使用无菌技术进行。强烈推荐使用外科助手在整个手术过程中保持不育。 </li><li>佩戴无菌手术手套，将开窗的无菌手术罩覆盖在动物上，将腿部暴露于中央开窗。 </li></ol> 5.进入生长板的外科手术_upload / 55571 / 55571fig1.jpg"/&gt; 图1:外科手术程序概述。 A）用于创建成功生长板损伤的几种解剖标记的位置。膝盖胶囊紧靠膝盖后面（白色），将胫骨与股骨分开。胫骨生长板（深红色）可以看到比膝盖骨更差，绕过胫骨。近端生长板是一个大致平面的平面，除了形成对角平面的前四分之一。这两个平面的交点形成生长板角度，用于适当的钻孔角度。半腱肌插入是在后胫骨插入四头肌肌肉的地方。 B）通过胫骨软组织的前内侧切口进入皮质骨。 C）使用与远端半腱插入物对齐的皮质窗口的位置作为参考点。 D）评估通过将牙科刀头上的斜面与皮质窗口对齐，损伤的深度。 <ol><li>使用＃3手术刀手柄和＃15刀片，从内侧股骨髁的远端开始沿着胫骨近端的内侧前方形成约1厘米的切口（ 图1A ）。 <ol><li>将皮肤紧紧靠在下面的骨头上，并在进行切口时牢固握住腿部。 注意:这将保持皮肤切口在所需的位置，并将有助于创建一个干净的切口。不要用手术刀太牢固，以避免穿刺膝盖胶囊，这将导致大量出血，并将使剩下的步骤变得困难。 </li></ol></li><li>记录重要的解剖标志物，包括:1）生长板，2）生长板角度，3）膝盖囊，4）半腱肌插入（ 图1A ）。 </li><li>使用手术刀，通过一个约0.5厘米的切口e筋膜和软组织在胫骨近端内侧，从生长板到皮肤切口底部（ 图1B ）。 </li><li>使用手术刀轻轻地从胫骨剥离或刮去筋膜和软组织（ 图1B ）。 注意:重要的是尽可能从胫骨去除或刮擦尽可能多的软组织，以免干扰钻孔步骤。 </li><li>在皮质透视下通过胫骨皮质骨穿刺皮质骨，用连接在旋转工具上的Steinmann针以10,000 RPM（材料部分规定的旋转工具的低速度）进行。创建皮质窗口，使其与远端半腱插入物对齐（ 图1C ）。 <ol><li>握住钻头垂直于胫骨骨干，慢慢钻孔，小心不要穿过骨干的另一侧;皮质窗口的深度只有〜2毫米，而不是感觉到阻力。 </li><li>如上所述，用另一只手紧紧握住腿。 注意:此步骤可能使用牙科钻头。然而，如果使用牙科钻头，腿部必须非常牢固地固定，以形成清洁的皮层窗口，并确保毛刺在所需位置处抓住并切割骨骼。鉴于其卓越的切割能力，Steinmann针脚被推荐用于此步骤。 </li></ol></li><li>用纱布戴皮质窗，如预期轻度出血。 </li></ol> 6.创造生长板损伤<ol><li>使用连接到旋转工具的1.8毫米牙科钻头通过中央生长板造成钻孔损伤。 注意:适当的深度，角度和方向对于破坏中心生长板是至关重要的（ 图1C和D ）。以下给出了获得适当深度，角度和方向的说明。 <ol><li>使用牙科钻头测量适当的深度，开始n通过将牙科钻头的端部与近端胫骨对准，半腱肌穿过膝盖囊（ 图1C ）。 </li><li>随着膝盖胶囊的牙齿结束，沿着半腱肌的毛刺轴，记下毛囊与皮质窗口对齐的位置。这是适当的深度，使钻头完全破坏生长板而不破坏关节面（ 图1C ）。 注意:牙科用于测量适当的深度。毛刺可以在与皮层窗口对齐的位置处用永久标记物标记，以在钻孔期间参考深度。然而，如果解剖标志物和上述方案被紧密参考，则这里规定的牙科钻头上的第一斜面（FG6）将适当地与皮质窗口对齐（ 如图1C所示 ）。 </li><li>要获得适当的钻头角度，请将旋转工具保持在较小的角度相对于胫骨骨干30度。 注意:这是一个视觉近似。 </li><li>为了获得适当的钻孔方向，瞄准生长板角度（ 图1C ）。沿着牙科钻头绘制视线到生长板角度以帮助产生中心缺陷。 </li><li>在进入皮质窗口之前，将旋转工具打开至10,000 RPM（材料部分指定的旋转工具的低速度）。 </li><li>旋转工具在适当的角度和方向，进入皮质窗口并推动旋转工具，直到毛刺标记与皮层窗口对齐。一旦达到适当的深度，取下旋转工具。 注意:以一个快速运动的方式进行生长板中断，使用生长板中的毛发最小化时间，以产生清洁的伤害。这对于数据分析很重要。 </li></ol></li><li>用纱布涂抹皮质窗口约30秒，因为预期出血。 </li><li>通过再次测量钻头长度来确保适当的伤害深度（步骤6.1.2）。 <ol><li>将钻头插入钻孔（旋转工具关闭），并将标记的钻头与皮质窗口对齐（ 图1D ）。 </li></ol></li><li>如果深度不足，请转动旋转工具并按压到所需的深度。 注意:虽然第二轮钻井并不理想，但完全破坏增长板块对于骨棒的发展至关重要。 </li><li>使用10 mL注射器和23号针头，用〜3 mL无菌盐水冲洗钻孔。 </li><li>用纱布干燥伤口。 </li></ol> 7.后伤程序<ol><li>如果评估基于生物材料的生长板处理，则使用适当尺寸的针（18至26号，取决于生物材料粘度）将生物材料通过钻孔注入损伤部位。 注意:生长板损伤的体积为〜3＆＃181; L，钻孔体积约20μL。可以注入生长板损伤和钻孔的材料的最大体积在20和25μL之间。 </li><li>通过用3-0聚乙醇酸缝线缝合筋膜来关闭伤口。在皮质窗口上涂抹骨蜡以分离下面的骨骼（可选）。 </li><li>用埋藏的缝合线或伤口夹闭合皮肤切口。 注意:建议使用伤口夹，因为动物会在伤口部位刮伤，并可能打开伤口。 </li><li>从异氟烷麻醉中取出动物，将其放在温暖的毯子上，并进行监测，直到它醒来。 </li><li>为了减少感染的风险，将动物放置在一个新的笼子里，用干燥的高压灭菌床垫。 </li><li>允许动物在手术后承担重量。 </li><li>手术后每12小时监测动物72小时，以检查感染的迹象，确保伤口夹固定在适当位置，并进行术后根据制度上认可的策略（ 例如，丁丙诺啡0.05mg / kg，每12小时36小时，卡洛芬5mg / kg，每24小时72小时）。 </li><li>在麻醉后10-14天取出伤口夹。 </li></ol>

<ol>
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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=Characterization+of+a+small+animal+growth+plate+injury+model+using+microcomputed+tomography.">Characterization of a small animal growth plate injury model using microcomputed tomography.</a> Bone. 46 (6), 1555-1563 (2010).</li><li>Lee, S. U., Lee, J. Y., Joo, S. Y., Lee, Y. S., Jeong, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+a+Scaffold-Free+Cartilage+Tissue+Analogue+for+the+Treatment+of+Physeal+Cartilage+Injury+of+the+Proximal+Tibia+in+Rabbits.">Transplantation of a Scaffold-Free Cartilage Tissue Analogue for the Treatment of Physeal Cartilage Injury of the Proximal Tibia in Rabbits.</a> Yonsei Med J. 57 (2), 441-448 (2016).</li><li>Planka, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotechnology+and+mesenchymal+stem+cells+with+chondrocytes+in+prevention+of+partial+growth+plate+arrest+in+pigs.">Nanotechnology and mesenchymal stem cells with chondrocytes in prevention of partial growth plate arrest in pigs.</a> Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 156 (2), 128-134 (2012).</li><li>Hansen, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-plate+chondrocyte+cultures+for+reimplantation+into+growth-plate+defects+in+sheep.+Characterization+of+cultures.">Growth-plate chondrocyte cultures for reimplantation into growth-plate defects in sheep. Characterization of cultures.</a> Clin Orthop Relat Res. (256), 286-298 (1990).</li><li>Cepela, D. J., Tartaglione, J. P., Dooley, T. P., Patel, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classifications+In+Brief:+Salter-Harris+Classification+of+Pediatric+Physeal+Fractures.">Classifications In Brief: Salter-Harris Classification of Pediatric Physeal Fractures.</a> Clin Orthop Relat Res. , (2016).</li><li>Salter, R. B., Harris, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injuries+Involving+the+Epiphyseal+Plate.">Injuries Involving the Epiphyseal Plate.</a> The Journal of Bone &amp; Joint Surgery. 83 (11), 1753 (2001).</li><li>Chung, R., Foster, B. K., Zannettino, A. C., Xian, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+roles+of+growth+factor+PDGF-BB+in+the+bony+repair+of+injured+growth+plate.">Potential roles of growth factor PDGF-BB in the bony repair of injured growth plate.</a> Bone. 44 (5), 878-885 (2009).</li><li>Fischerauer, E., Heidari, N., Neumayer, B., Deutsch, A., Weinberg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatial+and+temporal+expression+of+VEGF+and+its+receptors+1+and+2+in+post-traumatic+bone+bridge+formation+of+the+growth+plate.">The spatial and temporal expression of VEGF and its receptors 1 and 2 in post-traumatic bone bridge formation of the growth plate.</a> J Mol Histol. 42 (6), 513-522 (2011).</li><li>Chung, R., Cool, J. C., Scherer, M. A., Foster, B. K., Xian, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+neutrophil-mediated+inflammatory+response+in+the+bony+repair+of+injured+growth+plate+cartilage+in+young+rats.">Roles of neutrophil-mediated inflammatory response in the bony repair of injured growth plate cartilage in young rats.</a> J Leukoc Biol. 80 (6), 1272-1280 (2006).</li><li>Chung, R., 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=Roles+of+Wnt/beta-catenin+signalling+pathway+in+the+bony+repair+of+injured+growth+plate+cartilage+in+young+rats.">Roles of Wnt/beta-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats.</a> Bone. 52 (2), 651-658 (2013).</li><li>Zhou, F. H., Foster, B. K., Zhou, X. F., Cowin, A. J., Xian, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+mediates+p38+MAP+kinase+activation+and+negatively+regulates+bone+formation+at+the+injured+growth+plate+in+rats.">TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats.</a> J Bone Miner Res. 21 (7), 1075-1088 (2006).</li><li>Arasapam, G., Scherer, M., Cool, J. C., Foster, B. K., Xian, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+COX-2+and+iNOS+in+the+bony+repair+of+the+injured+growth+plate+cartilage.">Roles of COX-2 and iNOS in the bony repair of the injured growth plate cartilage.</a> J Cell Biochem. 99 (2), 450-461 (2006).</li></ol>

作者没有什么可以披露的。

增长板损伤动物模型大大增加了我们对这种损伤的生物学机制的理解，从而潜在地导致对患有生长板损伤的儿童的更有效的治疗干预。为了成功创建一个骨棒，并使用本工作中提出的模型研究其体内形成，关键是通过钻孔到足够的深度而不破坏关节软骨来破坏生长板。动物的手术实施变化，以及在较小程度上解剖标志物的变化可能会导致问题的结果。我们建议执行上述关于尸体动物?...

生长板损伤占所有儿科骨折的30％，可能导致骨生长受损1 。除骨折外，生长板受伤可能由其他病因引起，包括骨髓炎2 ，原发性骨肿瘤3 ，放射和化学疗法4以及医源性损伤5 。生长板（或Physis）是儿童长骨结束时的软骨区域，负责纵向骨生长。它通过软骨内骨化促进骨伸长;软骨细胞经历增殖和肥大，然后由进入的成骨细胞重塑以形成小梁骨6 。生长板也是发育骨架的弱区，容易受伤。生长板骨折或损伤的主要问题是生长板内损伤的软骨组织可以be替换为不需要的骨修复组织，也被称为"骨棒"。根据其生长盘中的大小和位置，骨棒可能导致角质畸形或完全生长停滞，对于尚未达到其全身高度的幼儿来说，这是一个破坏性的后遗症7 。 目前没有治疗可以完全修复受伤的生长板。一旦骨髓形成，临床医生必须决定是否手术切除它8 。至少2年或2厘米骨骼生长剩余的患者和跨越小于生长板面积的50％的骨棒通常是骨髓切除术的候选者8 。骨髓移植通常是随机插入自体脂肪移植物，以防止骨组织的重组，并允许周围的未受伤的生长板恢复生长。然而，这些技术是问题激素经常失败，导致骨条复发，对生长持续负面影响9 。迫切需要开发有效的治疗方法，不仅可以防止骨形成，还可以使生长板软骨再生，从而恢复正常的骨伸长。 骨棒形成的分子机制尚未完全阐明。对这些生物学机制的更多了解可以为受损生长板损伤的儿童带来更有效的治疗干预措施。由于在人类中研究这些机制是困难的，所以已经使用动物模型，特别是生长板损伤的大鼠模型10,11,12,13,14,15,16 。本文介绍的方法论文描述了大鼠胫骨生长板中的钻孔缺陷如何导致可预测和可重复的修复组织，其早于损伤后7天开始骨化，并且在损伤后28天形成完全成熟的骨块，重塑。这提供了一种小型动物体内模型，其中研究骨形成的生物学机制，以及评估可以预防骨棒和/或再生生长板软骨的新疗法。例如，该模型可用于测试可以再生生长板软骨的软骨形成生物材料，并为患有生长板损伤的儿童提供有价值的治疗。本文介绍的技术将描述用于产生生长板损伤的手术方法和随后将生物材料递送至损伤部位。我们还将讨论评估骨棒形成和修复组织的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>Scalpel handle</td>
			<td>McKesson</td>
			<td>MCK42332500</td>
			<td></td>
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			<td>Needle holder</td>
			<td>Stoelting</td>
			<td>RS-7824</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson tissue forceps</td>
			<td>Sklar</td>
			<td>50-3048</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors</td>
			<td>Sklar</td>
			<td>47-1246</td>
			<td></td>
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			<td>Rotary Tool</td>
			<td>Dremel</td>
			<td>7700</td>
			<td>Variable speed rotary tool&#160;</td>
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			<td>Keyless Rotary Tool Chuck</td>
			<td>Dremel</td>
			<td>4486</td>
			<td></td>
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			<td>Dental Burs</td>
			<td>Dental Burs USA</td>
			<td>FG6</td>
			<td>Round carbide bur, &#8804;2mm</td>
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			<td>Steinmann pins</td>
			<td>Simpex Medical</td>
			<td>T-078</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clippers</td>
			<td>Wahl&#160;</td>
			<td>5537N</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 PGA surutes</td>
			<td>Oasis</td>
			<td>MV-J398-V</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze 2x2&#34;</td>
			<td>Covidien</td>
			<td>441211</td>
			<td></td>
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		<tr>
			<td>Povidone Iodine</td>
			<td>McKesson</td>
			<td>922-00801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Vetone</td>
			<td>510224</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml luer lock syringe</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>23 gauge needle</td>
			<td>Becton Dickinson</td>
			<td>305145</td>
			<td></td>
		</tr>
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			<td>Isopropyl alcohol pads</td>
			<td>Dynarex</td>
			<td>1113</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>IsoFlo</td>
			<td>30125-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Caliper</td>
			<td>Mitutoyo</td>
			<td>500-196-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Rimadyl</td>
			<td>27180</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Par Pharmaceuticals Inc</td>
			<td>NDC 42023-179</td>
			<td></td>
		</tr>
		<tr>
			<td>Fenestrated Surgical Drape</td>
			<td>McKesson</td>
			<td>25-517</td>
			<td></td>
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		<tr>
			<td>Surgical Gloves</td>
			<td>Uline</td>
			<td>S-20204</td>
			<td></td>
		</tr>
		<tr>
			<td>#15 Scalpel Blade</td>
			<td>Aven</td>
			<td>44044</td>
			<td></td>
		</tr>
		<tr>
			<td>9mm wound clips</td>
			<td>Fine Science Tools</td>
			<td>12032-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex clip applier</td>
			<td>World Precision Instruments</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbant underpads</td>
			<td>McKesson</td>
			<td>MON 43723110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tec 3 Iso Vaporizer&#160;</td>
			<td>VetEquip</td>
			<td>911103&#160;</td>
			<td></td>
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		<tr>
			<td>Germinator 500</td>
			<td>Braintree Scientific</td>
			<td>GER&#160;5287-120V</td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water recirculator</td>
			<td>Kent Scientific</td>
			<td>TP-700</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent Underpads</td>
			<td>Medline Industries</td>
			<td>MSC281230</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat tibial growth plate injury model to characterize repair mechanisms and evaluate growth plate regeneration strategies

所有儿科骨折的三分之一涉及生长板，并可能导致骨生长受损。生长板（或分裂）是在所有长骨结束时发现的软骨组织，其负责纵向骨生长的儿童。一旦损伤，生长板内的软骨组织就可能发生过早的骨化，并导致不必要的骨修复组织，形成"骨棒"。在一些情况下，这种骨棒可能导致骨生长畸形，例如角度畸形，或者可以完全停止纵向骨生长。目前没有临床治疗可以完全修复受伤的生长板。使用生长板损伤的动物模型更好地了解骨棒形成的机制，并确定抑制它的方法是开发更好的生长板损伤治疗的好机会。该协议描述了如何使用钻孔缺陷来破坏大鼠近端胫骨生长板。这个sma动物模型可靠地产生一个骨棒，并可能导致类似于在儿童中看到的生长畸形。该模型允许调查骨条形成的分子机制，并且作为测试生长板损伤的潜在治疗选择的手段。

使用该方法成功的生长板损伤涉及胫骨生长板的中心的破坏而不破坏关节软骨表面。据报道，骨修复组织在损伤后约7天开始，并在损伤后28天完全发育，通过微型计算机断层扫描（micro CT）显现（ 图2 ）。虽然这些时间点在这里被选择以显示基于先前公布的数据的骨形成的开始和成熟，但是可以使用其他时间点来研究修复过程的各个阶段，从术后第1至6个月<sup...

Watch this Scientific Journal Video about A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies at JoVE.com

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

生长板是发生纵向生长的儿童长骨中的软骨区域。当受伤时，骨组织可能形成并损害生长。我们描述了生长板损伤的大鼠模型，其导致骨修复组织，允许研究修复机制和生长板再生策略。

大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

a-rat-tibial-growth-plate-injury-model-to-characterize-repair

Research

JoVE Journal

Medicine

11.0K 次观看.  University of Colorado Anschutz Medical Campus. 生长板是发生纵向生长的儿童长骨中的软骨区域。当受伤时，骨组织可能形成并损害生长。我们描述了生长板损伤的大鼠模型，其导致骨修复组织，允许研究修复机制和生长板再生策略。 

视频: A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

科研

医学

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

在家兔模型宫内生长受限胎儿超声心动图和多普勒超声脉冲波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

手术应激探索术后免疫抑制的机制和评估新围手术期免疫疗法的小鼠肿瘤模型

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Renal Ischaemia Reperfusion Injury: A Mouse Model of Injury and Regeneration

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable during renal transplantation. Experimental models that accurately and reproducibly recapitulate renal IRI are crucial in dissecting the pathophysiology of AKI and the development of novel therapeutic agents. Presented here is a mouse model of renal IRI that results in reproducible AKI. This is achieved by a midline laparotomy approach for the surgery with one incision allowing both a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia of the left kidney. By careful monitoring of the clamp position and body temperature during the period of ischaemia this model achieves reproducible functional and structural injury. Mice sacrificed 24 hr&#160;following surgery demonstrate loss of renal function with elevation of the serum or plasma creatinine level as well as structural kidney damage with acute tubular necrosis evident. Renal function improves and the acute tissue injury resolves during the course of 7 days following renal IRI such that this model may be used to study renal regeneration. This model of renal IRI has been utilized to study the molecular and cellular pathophysiology of AKI as well as analysis of the subsequent renal regeneration.

The mouse model of renal ischaemia reperfusion injury described here comprises of a right nephrectomy that provides control tissue and clamping of the left renal pedicle to induce ischaemia that results in acute kidney injury. This model uses a midline laparotomy approach with all steps performed via one incision.

肾缺血再灌注损伤:损伤的小鼠模型和再生

Renal ischaemia reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) in patients and occlusion of renal blood flow is unavoidable ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

补偿性增长龙鼠标肺切除型号

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

建立一个人类多发性骨髓瘤异种移植模型中的鸡，研究肿瘤的生长，侵袭和血管生成

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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 ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

大鼠颈动脉球囊损伤模型试验抗血管重建治疗

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

鼠屈肌腱损伤修复手术

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

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 ...