本研究由国家自然科学基金 82101000 （H. W.）、U21A20368 （L. Y.） 和 82100982 （F. L.） 资助，并由四川省科技计划 2023NSFSC1499（H. W.） 资助。

本研究所有动物手术均由四川大学华西口腔医学院伦理委员会（WCHSIRB-D-2021-597）审查批准。本研究使用Sprague-Dawley大鼠（雄性，体重300g）。
1. 术前准备
<ol><li>仪器准备<ol><li>本研究使用的手术器械见 图1A :电动剃须刀、组织剪刀、眼科剪刀、眼科镊子、一次性使用手术刀、骨膜分离器、口腔低速手机、口腔探头、一次性使用冲洗吸尘器、针架、3.0缝合线。</li>
		<li>准备一个口腔探针，并根据缺陷的直径用记号笔标记探针的大弯曲端（图1B）。使用它来确定手术过程中缺陷的大小。</li>
		<li>使用前对用于执行该程序的所有手术材料和器械进行消毒。将所需材料包装在折叠的布或包装纸中，并用高压灭菌胶带密封以进行蒸汽灭菌（125-135°C 20-25 分钟）。</li>
		<li>手术区域准备:喷洒75%酒精，对手术台和手术台周围环境进行消毒。在手术台上创建一个大约 60 cm x 90 cm 的无菌区域，用高压灭菌的窗帘。</li>
	</ol></li>
	<li>麻醉准备<ol><li>用10%盐酸氯胺酮（50-100mg / kg）和2%甲苯噻嗪2mg / kg腹膜内麻醉大鼠。使用皮下注射卡洛芬（5 mg/kg）进行术前和术中镇痛。通过脚趾捏试验检查麻醉深度。麻醉后，将无菌眼膏涂抹在眼睛上，以防止眼睛干涩和角膜损伤。</li>
	</ol></li>
</ol>2. 外科手术
<ol><li>将大鼠置于无菌手术台上的侧卧位置，并用电动剃须刀去除下肢毛发。</li>
	<li>使用5%的碘伏溶液和75%的酒精对手术区域的皮肤组织进行消毒。</li>
	<li>找到股骨近端和远端，沿股骨的长轴做一个2.5厘米的切口，切开大鼠的皮肤组织。<ol><li>用眼科镊子和组织剪刀将皮肤层与筋膜分开，并通过股二头肌和股外侧肌肉暴露到股骨的外侧通路。</li>
		<li>找到两块肌肉间隔的交叉点（一条白色组织线），并用一次性手术刀片沿着肌肉边界小心地分开，直到到达股骨表面。 注意:使用一次性刀片分离肌肉时，重要的是沿着肌肉隔膜分离，并注意在软组织内引起血管损伤。初学者和不熟悉解剖结构的人必须使用眼科钳子和骨膜分离器在两个肌肉块之间进行钝性解剖。</li>
	</ol></li>
	<li>应用骨膜分离器将股骨表面肌肉钝性分离，并暴露股骨干中部。</li>
	<li>使用无菌记号笔标记位于股骨头外侧 1/3 斜嵴顶部的股骨干中表面的缺损部位区域。</li>
	<li>使用口腔低速手机搭配直径为 1.2 毫米的慢动作球钻，在标记部位垂直于骨表面钻一个小孔，破坏骨膜和骨皮质，深度足以到达骨髓腔。在此钻孔深度水平上，向所有方向扩展平行于该深度的孔的尺寸，修整以实现箱形腔形状。</li>
	<li>使用平行于缺陷边缘的标记口腔探针来确定制备过程中和之后的缺陷直径和形态。</li>
	<li>分别用 3-0 条单丝可吸收缝合线闭合肌肉层和皮肤层，并从内到外用 5% 的碘伏对手术区域进行消毒。</li>
</ol>3. 术后护理
<ol><li>手术后，皮下给予卡洛芬（5mg / kg），并将大鼠置于恒温加热垫上，直至从麻醉中恢复。当老鼠恢复意识时，轻轻地将其移至装有干燥高压灭菌垫料的笼子中。</li>
	<li>术后继续镇痛 24 小时，术后监测 1 周。</li>
</ol>4. 样品采集和分析
<ol><li>手术后通过腹膜内注射戊巴比妥钠100-200mg / kg对大鼠进行人性安乐死。小心地分离股骨表面的肌肉和筋膜组织，并完全切除手术侧的股骨。术后0天（图2A，B），2周，4周和6周收集标本。</li>
	<li>将股骨标本固定在4%多聚甲醛中24小时。使用显微计算机断层扫描分析股骨的结构。设置扫描参数如下:X射线管电位，70 kVp;过滤器，AL 0.5 mm;X 射线强度，0.2 mA;体素尺寸，17 μm;积分时间为 1 × 300 ms。使用位图数据重建 3D 模型图像。</li>
	<li>在10%EDTA中将标本脱钙8周，然后在一系列分级乙醇稀释液中对股骨标本进行脱水。然后，将样品嵌入石蜡15 中。<ol><li>将嵌入的样品从矢状面切成5μm石蜡切片。</li>
		<li>使用苏木精和伊红 （H&E） 染色试剂盒和 Masson 染色试剂盒对切片进行染色。通过组织病理学观察缺损区域的愈合情况。</li>
	</ol></li>
</ol>

大鼠股骨盒的建立和评估:空腔缺陷模型

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所有原始数据和图像均包含在本文中。作者声明没有利益冲突

临床前动物模型对于检查骨愈合和生物材料对骨再生的影响至关重要。该协议说明了股骨箱腔缺损模型，该模型复制了与临床骨再生相关的膜内骨形成过程。使用预先标记的口腔探针对缺陷区域进行术中标准化。显微 CT 和组织病理学染色结果显示，6 周内逐渐愈合，骨膜增厚，新小梁骨形成，随后皮质骨形成致密。在愈合过程中未观察到软骨组织，表明膜内成骨。这一发现为研究生物材料或药物在临床骨再生中的作用提供了新的视角。
该协议需要特别注意几个因素:（i） 深度麻醉是防止动物痛苦和确保准确定位缺陷的先决条件。应警惕监测麻醉深度和生命体征。（ii）在肌肉分离过程中，应注意防止血管损伤，以免阻碍恢复。（iii） 在准备缺陷孔时，应保持右侧支点稳定并始终垂直于骨表面，以确保箱形缺陷处于相同的深度。应控制钻孔深度，以去除皮质骨组织，但又不能过深。这避免了由于骨髓的过度机械破坏而导致的骨愈合延迟，从而导致间充质干细胞 （MSCs） 的去除和炎症反应的破坏16。（iv） 龋洞准备需要助手不断用生理盐水冲洗该区域，以减轻骨消融过程中由热量造成的潜在组织损伤，同时保持手术区域的可见性。
骨折、节段性骨缺损和颅骨缺损模型是研究骨再生中常用的动物模型。骨折模型反映了创伤的实际过程，但由于实验动物之间的骨折横截面差异，结果可能会有所不同17.节段性骨缺损模型可有效模拟骨折并发症，例如骨不连或愈合延迟。由于其解剖学定位，该模型有助于通过放射学或组织学方法评估新骨的形成和血运重建18。然而，这种方法在受试者之间的缺陷大小存在相当大的差异;夹板固定或金属植入物内固定的要求也限制了其应用11.颅骨缺损模型的特点是没有内固定，尺寸和解剖位置的可重复性高，以及稳定的机械环境14，这也限制了在评估植入材料对生理、生物力学负荷的生物反应方面的应用13。此外，在没有移植骨的情况下，硬脑膜和覆盖的软组织对缺损的侵入会阻碍骨再生，使这些模型对支架材料的性能敏感18。因此，该协议建议在大鼠股骨干中建立箱腔缺损，其允许使用简单的工具18在术中测量缺损直径。与断裂或节段性缺陷模型相比，这种方法可以更好地控制缺陷标准化，并且无需进行内部或外部固定。此外，该模型对由于软组织侵蚀导致的骨形成损伤具有鲁棒性。然而，该协议存在某些局限性:（i） 执行不当或缺陷直径过大会导致大鼠严重出血、休克甚至死亡。因此，为了确保动物的安全和福利，在对活体受试者进行手术之前，必须进行老鼠尸体的处理。（ii） 此外，啮齿动物皮层19 中缺乏 Haversian 系统，因此很难完全再现人类骨折/缺损的自然临床愈合过程。大鼠作为啮齿动物，具有成本效益，需要最少的维护，并且安全且易于处理，比兔子、狗和山羊等大型动物具有更高的可重复性20。
综上所述，我们建立了大鼠股骨干箱腔缺损模型，并利用显微CT和组织病理学染色技术跟踪愈合过程。该协议提供了一种全面且可推广的方法，用于在具有临床意义的骨再生模型中检查膜内骨形成。此外，本文还为替代生物材料、骨再生相关药物、支架等的生物学性能评价提出了创新视角。它还有助于骨组织工程的紧急治疗策略的临床前验证。

骨折和有缺陷的骨头通常由外伤、肿瘤、炎症和先天性畸形引起 1,2。尽管年轻健康个体的骨组织通常具有强大的再生能力3，但超过临界尺寸的缺陷或由于全身性疾病（例如糖尿病、骨质疏松症和感染）导致的愈合障碍仍可能导致并发症，例如骨骼不连续性或愈合受损4。为了应对这一临床挑战，骨移植或生物材料通常用于替换严重有缺陷的骨骼或重建大骨段。然而，这些治疗有局限性。例如，尽管被认为是金标准，但自体骨移植受到供体供应限制和潜在供体部位并发症的影响 5,6。同种异体移植物也存在一定的风险，例如免疫介导的排斥反应、疾病的潜在传播以及对移植物的生物力学和生物学特性的负面影响7.
近年来，专注于骨缺损愈合机制的研究激增。替代生物材料的使用和组织工程的进步已成为骨再生领域的突出话题8.在这些生物材料可以应用于人体治疗之前，它们必须在体外和体内进行测试，以确保其有效性和安全性。然而，体外环境复杂性的降低以及免疫和炎症反应的缺乏限制了各种生物材料的体外评估。因此，需要为各种类型的骨组织缺损建立动物模型9.动物模型允许在不同负载条件下评估生物材料，促进对物种特异性骨骼特征的理解，并深入了解动物模型与人类临床情况之间的相似性。这些优势对于研究骨-支架相互作用并将研究结果转化为临床实践至关重要 9,10。
目前，机械骨缺损动物模型被广泛用于验证生物材料的性能，其中颅骨缺损模型和节段性骨缺损模型是最常用的方法11。节段性骨缺损模型通常用于模拟以骨不连结束的严重长骨或胫骨创伤，由于其均匀的尺寸和明确的解剖位置而具有优势，简化了新骨形成和血运重建的放射学或组织学评估。然而，这些模型需要金属植入物来稳定双侧骨折段，并且需要涉及软骨内和膜内骨化的复杂愈合过程12。另一方面，颅骨缺损模型因其标准化的缺损直径、方便的手术通路以及硬脑膜和软组织的支持功能而成为评估生物材料的主要筛选工具13。尽管它们被广泛用于在临床相关场景中模拟膜内骨形成，但由于它们在愈合过程中的非承重性质，它们不适合评估生物力学负荷条件下的骨愈合14。
为了解决这些局限性，我们在大鼠股骨干组织中建立了箱腔皮质骨缺损模型。利用显微计算机断层扫描（CT）、三维（3D）重建和组织病理学染色（苏木精和伊红[HE]和Masson），分析了该模型在止血条件下的愈合过程。我们旨在为评估生物材料在生物力学负载条件下的性能以及研究骨再生相对于膜内骨化的生物工程和机制提供新的见解。

<table><tbody><tr><td>1.2 mm slow speed ball drill</td><td>Dreybird Medical Equipment Co., Ltd.</td><td>RA3-012</td><td>For preparation of box cavity defects</td></tr><tr><td>3.0 suture</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td>For suturing wounds</td></tr><tr><td>4% paraformaldehyde</td><td>Biosharp</td><td>BL539A</td><td>For fix the femoral specimens</td></tr><tr><td>Cotton balls</td><td>Haishi Hainuo Group Co.,&amp;nbsp; Ltd.</td><td>20120047</td><td>For skin sterilization and cleaning of surgical field</td></tr><tr><td>Cotton sticks</td><td>Lakong Medical Devices Co., Ltd.</td><td>M6500R</td><td>For skin disinfection</td></tr><tr><td>Dental technician grinding machine</td><td>Marathon</td><td>N3-140232</td><td>For preparation of box cavity defects</td></tr><tr><td>Disposable scalpel</td><td>Hangzhou Huawei Medical Supplies Co., Ltd.</td><td>20100227</td><td>For creating skin incisions as well as to sharply separate muscle tissue</td></tr><tr><td>Electric shaver</td><td>JASE</td><td>BM320210</td><td>Removal of hair tissue from the surgical area</td></tr><tr><td>Hematoxylin and Eosin Stain kit</td><td>Biosharp</td><td>C1005</td><td>For the histological analysis of the specimens</td></tr><tr><td>Masson&amp;rsquo;s Trichrome Stain Kit</td><td>Solarbio</td><td>G1340</td><td>For the histological analysis of the specimens</td></tr><tr><td>Micro CT</td><td>Scanco medical ag</td><td>&amp;micro;CT 45</td><td>For analyzing the healing of defects in femoral samples</td></tr><tr><td>Needle holder</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td>For suture-holding needles</td></tr><tr><td>Olympus Research Grade Whole Slide Scanning System VS200</td><td>Chengdu Knowledge Technology Co.</td><td>VS200</td><td>For analyzing the results of HE staining and Masson staining</td></tr><tr><td>Ophthalmic forceps</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td>For clamping skin, muscle tissue</td></tr><tr><td>Ophthalmic scissors</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td>For forming a skin incision approach</td></tr><tr><td>Oral low-speed handpiece</td><td>Marathon</td><td>Y221101003</td><td>For preparation of box cavity defects</td></tr><tr><td>Oral probe</td><td>Shanghai Sangda Medical Insurance Co., Ltd.</td><td>20000143</td><td>For measuring the diameter of defects</td></tr><tr><td>Periosteal separator</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td>For blunt separation of muscle tissue</td></tr><tr><td>Sprague&amp;ndash;Dawley rats</td><td>Byrness Weil Biotech Ltd</td><td>None</td><td>For the establishment of femoral bone boxy cavitary defect</td></tr><tr><td>Tissue scissors</td><td>Chengdu Shifeng Co., Ltd.</td><td>None</td><td>For forming a skin incision approach</td></tr></tbody></table>

creating a box-cavity defect model in the cortical bone of rat femora

严重的骨缺损或复杂骨折会导致严重的并发症，例如骨不连或骨愈合不足。组织工程涉及细胞、支架和细胞因子的应用，被认为是骨再生的有前途的解决方案。因此，模拟骨骼缺陷的各种动物模型在探索组织工程治疗骨愈合的潜力方面发挥着至关重要的作用。本研究建立了大鼠股骨中部的箱形皮质骨缺损模型，可作为评估生物材料促进骨愈合功能的理想模型。这种箱形皮质骨缺损是使用口腔低速手机钻孔的，并通过车床针进行成型。术后立即进行显微CT分析，确认箱腔皮质骨缺损成功建立。然后在手术后的多个时间点（0 天、2 周、4 周和 6 周）收获大鼠手术侧的股骨。使用micro-CT，苏木精和伊红（H&E）染色以及Masson三色染色评估了每个样品缺陷区域的愈合过程。这些结果表明，愈合模式与膜内骨化一致，6 周内基本愈合完成。这种动物模型的愈合过程的分类提供了一种有效的 体内 方法，用于研究在骨组织缺损愈合过程中靶向膜内骨化的新型生物材料和药物。

在该协议中，我们通过钻孔成功建立了尺寸为4.5 mm x 1.5 mm的大鼠股骨箱腔缺陷模型。为了分析手术侧的愈合过程，我们在术后0 d、2周、4周和6周采集了手术侧股组织，这是大鼠股骨创伤愈合过程中软骨内骨化、膜内骨化和骨重塑的关键时间点2。在术后第 0 天，从显微 CT 位图数据重建 3D 模型显示，我们成功模拟了股骨中的箱腔缺损，其尺寸为 4.5 mm x 1.5 mm 深到骨髓腔（图 3A）。显微 CT 结果显示，在模型创建后 2 周，皮质骨缺损的间隙中矿化小梁骨形成（图 3B）。缺损区域的新骨组织表面显示出成熟、致密的皮质骨，术后 4 周髓侧仍可见小梁骨组织（图 3C）。6周后，缺损区域几乎由成熟、致密的皮质骨组成，髓侧附近仅留下少量小梁骨组织，表明缺损区域已基本愈合（图3D）。
我们还通过H&E染色和Masson三色染色分析收集的标本（图4）。结果显示，术后2周，缺损区形成幼稚小梁样骨组织，缺损区周围骨膜增厚，与缺损区新生小梁骨组织相连（图4A，A'，B，B'）。在产生缺损后4周，在缺损区域表面形成致密的皮质骨组织，并与缺损两侧的皮质骨相连;小梁样骨组织在髓侧被严重吸收（图4C，C'，D，D'）。在缺损区域形成成熟的皮质骨组织，术后6周髓侧的小梁骨组织几乎完全吸收（图4E，E'，F，F'），表明缺损的愈合过程几乎完成。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66068/66068fig1v2.jpg"> 图 1:创建缺陷所需的仪器。 （A） 手术器械。（1）电动剃须刀;（2）眼科钳;（3）眼科剪刀;（4）一次性使用手术刀;（5）骨膜分离器;（6）口腔低速手机;（7）改良口服探针;（8）一次性使用喷灌真空吸尘器;（9）针架;（10）组织剪刀;（11）3.0缝合线。（B） 标有缺陷直径尺寸的口腔探针。在无菌一次性口服探针的大弯曲端用 1.5 mm 和 4.5 mm 刻度标记，用于术中测量缺损区域的直径。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66068/66068fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66068/66068fig2v2.jpg"> 图 2: 箱腔缺陷的形态（第 0 天）。 （A，B） 使用用于创建箱腔缺陷 （4.5 mm x 1.2 mm） 的改良口腔探针测量第 0 天手术后样本。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66068/66068fig2v2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66068/66068fig3v3.jpg"> 图 3:缺损区域愈合过程的 Micro-CT 分析。 术后 0 天、14 天、28 天和 42 天的缺损区域 3D 重建图像。（A） 纵向平面 .（B-D）14 天、28 天和 42 天时缺损区域愈合的纵向（左图）、矢状面（中图）和横截面（右图）显微 CT 图像。A、B、C 和 D 中的黄色虚线框代表缺陷区域。黄色虚线代表缺损的边缘，黄色箭头代表缺损区域中的新骨组织。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66068/66068fig3large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66068/66068fig4v2.jpg"> 图4.组织病理学结果。 （A-F）14 天、28 天和 42 天对缺陷区域进行 H&E 和 Masson 染色。（A'-F'）A-F 中显示的黑色矩形框的放大图像。术后14 d在缺损区域形成小梁样矿化骨组织;术后28 d，在缺损区域表面形成皮质骨组织，并与缺损区域的两端相连;术后42 d，缺损区域形成成熟的皮质骨组织，愈合基本完成。蓝色虚线表示缺陷的边缘;用红色虚线圈出的区域是缺损区域的新骨组织;黑色箭头是增厚的骨膜;黄色三角形是旧的皮质骨;绿色箭头是新的小梁状骨组织;黑色和黄色的星星是新的皮质骨组织;红色箭头是新的骨膜。比例尺:500 μm （A-F）、200 μm （A'-F'）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66068/66068fig4large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora at JoVE.com

作者聚焦:用于增强生物材料和骨再生评估的箱腔皮质方法

在这里，我们提出了一种在大鼠股骨干组织中产生箱腔缺损的方案。该模型可以评估生物材料在生物力学应力下的性能，并探索与膜内成骨相关的骨再生机制。

在大鼠股骨皮质骨中创建箱腔缺损模型

Severe bone defects or complex fractures can result in serious complications such as nonunion or insufficient bone healing. Tissue engineering, which ...

各种具有骨缺损的动物模型被应用于探索组织工程对骨愈合的治疗潜力。然而，它们中的大多数在生物力学负载条件下无法产生标准缺陷，这对于评估生物材料的骨愈合效果具有重要意义。与传统的骨缺损模型相比，最近的箱腔皮质骨缺损模型提供了显着的进步。它不仅可以提供标准化的骨缺损尺寸，以便更准确地量化骨愈合，而且还允许在生物力学负载条件下评估生物材料，而无需额外固定。我们的方案提供了一种全面且可推广的方法，用于在骨再生模型的体内研究中研究膜内骨形成。这对骨组织工程领域特别有益。

creating-a-box-cavity-defect-model-in-the-cortical-bone-of-rat-femora

Nanyang Technological University

Research

JoVE Journal

Medicine

Creating a Box-Cavity Defect Model in the Cortical Bone of Rat Femora

622 次观看.  West China Hospital of Stomatology, Sichuan University. 各种具有骨缺损的动物模型被应用于探索组织工程对骨愈合的治疗潜力。然而，它们中的大多数在生物力学负载条件下无法产生标准缺陷，这对于评估生物材料的骨愈合效果具有重要意义。与传统的骨缺损模型相比，最近的箱腔皮质骨缺损模型提供了显着的进步。它不仅可以提供标准化的骨缺损尺寸，以便更准确地量化骨愈合，而且还允许在生物力学负载条件下评估生?...

视频: 作者聚焦:用于增强生物材料和骨再生评估的箱腔皮质方法

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 ...

科研

医学

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (&#181;CT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent &#181;CT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

The goal of this protocol is to generate a nude rat osteoporosis-related vertebral compression fracture model that can be longitudinally evaluated in vivo using a semiautomated microcomputed tomography-based quantitative structural analysis.

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

生物工程

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

大鼠牵张成骨的高效可再生性协议

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

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 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. ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

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 ...

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 ...

<meta charset="utf8"></head><body>用于再生研究的标准化大鼠颅骨缝合骨缺损模型

The Establishment of Calvarial Suture-Bony Composite Defects in Rats: A Standardized Model for Suture-Regenerative Therapy Investigation

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development (the latter occurs in the developing cranium). However, the lack of a standardized model hinders progress in investigating suture-regenerative therapies and poses challenges for conducting comparative analyses across distinct studies. To address this issue, the current protocol describes the detailed modeling process of calvarial suture-bony composite defects in rats.
The model was generated by drilling full-thickness rectangular holes measuring 4.5 mm &#215; 2 mm across the coronal sutures. The rats were euthanized, and the cranium samples were harvested postoperatively at day 0, week 2, week 6, and week 12. &#181;CT results from samples collected immediately post-surgery confirmed the successful establishment of the suture-bony composite defect, involving the removal of the coronal suture and the adjacent bone tissues.
Data from the 6th and 12th postoperative weeks demonstrated a natural healing tendency for the defect to close. Histological staining further validated this trend by showing increased mineralized fibers and new bone at the defect center. These findings indicate progressive suture fusion over time following calvarial defects, underscoring the significance of therapeutic interventions for suture regeneration. We anticipate that this protocol will facilitate the development of suture-regenerative therapies, offering fresh insights into the functional restoration of calvarial defects and reducing adverse outcomes associated with suture loss.

<meta charset="utf8"></head><body>作者聚焦:颅骨缝合骨复合缺陷标准化大鼠模型的开发和评估

We describe the detailed surgical procedures of calvarial suture-bony composite defects in rats, alongside the investigations into the short-term and long-term prognoses of the model. We aim to construct a standardized model for developing suture-regenerative therapies.

大鼠颅骨缝合骨复合缺损的建立:缝合再生治疗研究的标准化模型

Large-scale calvarial defects often coincide with cranial suture disruption, leading to impairments in calvarial defect restoration and skull development ...

在大鼠股骨皮质骨中创建箱腔缺损模型

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半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

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在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

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

全层软骨缺损大鼠模型的开发与评价

大鼠颅骨缝合骨复合缺损的建立:缝合再生治疗研究的标准化模型

大鼠颅骨缝合骨复合缺损的建立:缝合再生治疗研究的标准化模型