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

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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. 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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的程序，以确保在进行活动动物研究的程序之前成功生长板损伤。虽然尸体动物缺乏组织柔韧性，不会流血，但这些动物的生长板损伤程序和解剖特征与活体动物相似。 Furt睾丸，可以容易地解剖尸体胫骨生长板，因为骨through通过施加轻力与干ysis分离，并且可以观察钻孔的位置。这种快速分析允许技术修改，以了解对尸体动物的适当的钻孔深度和角度，而不需要成像。 应该指出，存在其他生长板损伤的动物模型。已经在小鼠中进行了类似的透孔缺陷，并导致骨形成20 。尽管其尺寸较小，但也可用于研究骨棒形成所涉及的机制。 Coleman 等人报道了另一种有效的生长板损伤大鼠模型，其中通过穿过关节软骨21钻出在股骨远端产生中心性透视缺损。这种方法也导致形成一个骨棒和肢体长度不平等，如同这里提供的模型。生长板损伤和治疗的其他动物模型包括兔22 ，猪23和羊24 。虽然较大的动物损伤模型可能更密切地代表临床损伤，但是大鼠模型对于植入物损伤的生物学机制的研究是有用的。例如，本文提出的大鼠模型已被广泛用于研究植骨损伤和骨形成过程10,11,12,13,14,15,16的分子机制。此外，大鼠模型可以用于在移动到更大的动物模型之前测试各种植物治疗。然而，这种大鼠生长板损伤模型的挑战是在骨骼内进行钻孔，国王不可能观察钻孔位于生长板内的位置。因此，活体动物生长板的成功破坏只能在手术时使用成像技术或通过在手术后7至28天评估骨棒形成来确认。通过实践，可以实现获得骨形成形成的高度成功，但早期研究可导致许多动物由于未受伤的生长板或生长不足而导致骨形成障碍盘子。 该模型的另一个限制是钻孔损伤不代表儿童正常的生长板损伤，这通常是由于骨折而发生的。生长板内的骨折可以使用Salter-Harris分类系统26进行分类。 III型和IV型生长板骨折最常导致导致的植骨损伤到骨棒形成。这里提出的生长板损伤类型最为密切地涉及VI型生长板损伤，这是一种罕见的损伤类型，其中通过创伤或穿刺伤口除去了分离物。然而，由于生长板损伤后骨形成的病理生理机制仍然难以捉摸，所以大鼠模型对发现这一过程仍然很重要，以便为患有各种生长板损伤的儿童开发新的治疗方案。这里描述的方法可靠地产生一个骨棒，可用于研究体内生长板损伤修复过程的多个方面17,27,28,29,30,31,32 。还显示，这种大鼠模型导致生长板后胫骨生长减少陪审团13 ，这使得它是一个更有趣的动物模型来测试导致生长板再生和潜在恢复骨伸长的新型治疗选择。 总之，本文详细介绍了创建生长板损伤模型的方法，通过该模型调查骨形成和体内生长板损伤的潜在治疗。鉴于骨生长板损伤后28天骨块完全成熟，该大鼠模型允许相对便宜且快速的研究。除了发现我们对体内骨棒形成的分子机制的理解，该模型可用于测试抑制骨形成形成并促进生长板软骨再生的生物材料。

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><tbody><tr><td>Scalpel handle</td><td>McKesson</td><td>MCK42332500</td><td></td></tr><tr><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></tr><tr><td>Rotary Tool</td><td>Dremel</td><td>7700</td><td>Variable speed rotary tool&amp;nbsp;</td></tr><tr><td>Keyless Rotary Tool Chuck</td><td>Dremel</td><td>4486</td><td></td></tr><tr><td>Dental Burs</td><td>Dental Burs USA</td><td>FG6</td><td>Round carbide bur, &amp;le;2mm</td></tr><tr><td>Steinmann pins</td><td>Simpex Medical</td><td>T-078</td><td></td></tr><tr><td>Hair clippers</td><td>Wahl&amp;nbsp;</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 2 x 2&quot;</td><td>Covidien</td><td>441211</td><td></td></tr><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><tr><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></tr><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>9 mm 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&amp;nbsp;</td><td>VetEquip</td><td>911103&amp;nbsp;</td><td></td></tr><tr><td>Germinator 500</td><td>Braintree Scientific</td><td>GER&amp;nbsp;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17 。 表1通过提供（1）在整个生长板内的骨体积分数和（2）bo的三次独立运行后28天的外科手术损伤的大鼠生长板中的骨量形成的概述修复组织区域内的体积分数仅为15 。数据报告为平均百分比±标准偏差，并表明在独立运行之间获得了类似的结果。通过单因素方差分析（ANOVA）分析不同运行之间的差异，运行之间没有统计学差异，表明模型的重现性。使用具有橙色G /曙红复色素18的阿尔辛蓝苏木精（ABH）组织学显示不同阶段的骨形成条件下的多种修复组织（ 图2 ）。使用这种组织学染色，可以鉴别和量化不同类型的修复组织，包括间充质，软骨，骨小梁和骨髓。 由于错误地遵循上述步骤可能会导致一些问题。不方便<html>钻头深度不会破坏生长板，这将导致很少或没有骨棒形成。关节软骨表面的破坏产生较大的损伤，可引起关节软骨进入生长板损伤部位，使愈合过程复杂化（ 图3A ）。以不适当的角度或方向破坏生长板会导致非中心损伤（ 图3B ）。在这种情况下，仍然会发生骨棒形成，尽管它将是所需位置的侧向或内侧。总体来说，修复生长板损伤后的组织可能以多种方式进行分析，包括microCT，定量PCR，组织学染色和免疫组织化学。除了组织学和分子测量之外，肢体长度和生长板测量提供了全骨增长的重要测量。据报受影响的肢体与未受伤的对照肢体相比经历生长减缓&gt; 13。在整个研究过程中，可以在不同的时间点测量肢体长度，使用microCT图像来研究肢体长度差异。以前使用的时间点的例子包括伤后28天和56天。生长板测量，包括总体高度，分区高度和系链形成也可以提供关于组织修复过程13,14,15的重要信息。理想情况下，应在手术前采取肢体长度和生长板测量以获得基线值。为了进一步阐明生物学机制或测试治疗的疗效，应设计适当的对照组，并且包括不受影响的四肢和四肢进行手术但未经治疗。 也可以在生长板损伤模型中测试生物材料。作为一个例子，一个i如步骤7.1所述，将山体微凝胶19注射到生长板损伤部位，并且在图4的损伤部位清楚地看到。随后的分析可能涉及确定生物材料对修复组织组成，肢体长度和生长板测量的影响，如前所述。 <img alt="图2" src="/files/ftp_upload/55571/55571fig2.jpg" /> 图2.成功生长板破碎和骨棒形成。 在微创CT后7天观察到骨形成，并通过阿尔辛蓝苏木精（ABH）染色证实。损伤后第28天，骨块完全成熟，如microCT和ABH染色所见。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55571/55571fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图3.不正确钻孔的潜在结果。 A）通过胫骨钻孔太远可能会破坏关节面，这会使愈合过程复杂化，并可能导致不确定的结果。 B）钻头的角度不正确会导致非中心生长板损伤。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55571/55571fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55571/55571fig4.jpg" /> 图4.用生物材料治疗生长板损伤。 ABH染色显示受损生长板中的壳聚糖微凝胶。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 公 </stroNG&gt; </td><td> 运行1 </td><td> 运行2 </td><td> 运行3 </td><td> P值 </td></tr><tr><td>整个生长板内骨体积分数</td><td> 9.76 +/- 3.81％ </td><td> 10.52 +/- 4.06％ </td><td> 11.93 +/- 2.04％ </td><td> 0.5493 </td></tr><tr><td>修复组织区域内的骨体积分数</td><td> 41.5 +/- 8.33％ </td><td> 46.08 +/- 10.12％ </td><td> 46.77 +/- 8.14％ </td><td> 0.5128 </td></tr></tbody></table> 骨量分数数据。 来自三个独立运行的未经治疗的大鼠的损伤后28天的数据来自微CT图像。

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

Nanyang Technological University

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

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

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

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

建立评估膜内骨化硬性稳定骨折，牵张成骨，或临界中小企业缺损愈合

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

医学

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

发育生物学

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

The tibial fracture-pin model is a clinically relevant model of orthopedic trauma comprising a unilateral open tibial fracture with intramedullary nail internal fixation and simultaneous injury to the tibialis anterior muscle. Thermal sensitivity in this model can be measured using a 45 s hot plate paradigm.

The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue ...

神经科学

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

全层软骨缺损大鼠模型的开发与评价

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

使用三系荧光小鼠的精确生长板损伤研究

Tricolor Transgenic Murine Model for Studying Growth Plate Injury

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones are overloaded.&#160;Better treatments for severely fractured growth plates are needed because the response to injury is a bony bridge that prematurely fuses the growth plate, leading to stunted and/or crooked limbs. Murine models of growth plate injury are advantageous for mechanistic studies, but&#160;are challenging because it is difficult to visualize and precisely injure the small growth plates in young mice.&#160;We describe here an improved growth plate injury model using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X.
These mice show native fluorescence associated with the three primary substrata of the growth plate. A growth plate injury similar to a Salter-Harris Type II injury is created reproducibly with a bur using the hypertrophic section of the growth plate as a reference during live imaging under fluorescence stereo microscopy guidance. Frozen histology analysis of the native fluorescence simplifies assessing the cellular response to injury. This methodology represents a substantial leap in growth plate injury research, providing a detailed and reproducible method for investigating pathology and evaluating new therapeutic strategies.

作者聚焦:在小鼠中使用三系荧光报告基因推进生长板损伤模型

This protocol describes an improved mouse model for adolescent bone growth plate injuries. Using transgenic mice with tri-lineage fluorescent reporters for collagen types I, II, and X, the primary matrices associated with three different substrata of the growth plate, injury placement is guided by native fluorescence under the microscope.

用于研究生长板损伤的 Tricolor 转基因小鼠模型

The cartilage growth plates in the bones of children enable limb lengthening but are weak relative to the bone, making them prone to fracturing when bones ...

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

摘要

探索更多视频

此视频中的章节

人类血管周围干细胞用于骨再生

建立评估膜内骨化硬性稳定骨折，牵张成骨，或临界中小企业缺损愈合

无胸腺大鼠模型工程前交叉韧带移植评估

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

大鼠双侧髌腱损伤模型中干细胞治疗的评价

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

全层软骨缺损大鼠模型的开发与评价

用于研究生长板损伤的 Tricolor 转基因小鼠模型

用于研究生长板损伤的 Tricolor 转基因小鼠模型

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

摘要

探索更多视频

此视频中的章节

人类血管周围干细胞用于骨再生

建立评估膜内骨化硬性稳定骨折，牵张成骨，或临界中小企业缺损愈合

无胸​​腺大鼠模型工程前交叉韧带移植评估

成年小鼠数字截肢和再生:研究哺乳动物暴发体形成和内膜性细胞化的简单模型

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

大鼠双侧髌腱损伤模型中干细胞治疗的评价

The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury

全层软骨缺损大鼠模型的开发与评价

用于研究生长板损伤的 Tricolor 转基因小鼠模型

用于研究生长板损伤的 Tricolor 转基因小鼠模型

无胸腺大鼠模型工程前交叉韧带移植评估