Name: in vivo evaluation of fracture callus development during bone healing in mice using an mri-compatible osteosynthesis device for the mouse femur
Uploaded: 2017-11-14T15:00:00.000+00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur at JoVE.com

我们感谢 Sevil Essig, 孙燕姿 Schroth, Verena 费舍尔, 卡嘉 Prystaz, 伊冯 Hägele 和安妮 Subgang 的优秀技术支持。我们还感谢德国研究基金会 (CRC1149, INST40/499-1) 和 AO 创伤基金会德国资助这项研究。

所有动物实验都遵守了对实验室动物的照顾和使用的国际规定, 并得到了区域管理当局的批准 (No. 1250、Regierungspr 和 #228; sidium T & #252; 宾, 德国)。所有小鼠每笼保持两到五只动物, 每个笼子上的 14 h 光, 10 小时黑暗的昼夜节律与水和食物提供了 ad 随意 . 
 1. 小鼠的外科材料和前处理的准备 <ol> <li> 对所有的外科材料进行消毒。使用灭菌温度为120-135 和 #176; C 用于20-30 分钟的灭菌时间. </li>
	<li> 从19-35 克体重之间的另一株 C57BL/6 中购买小鼠或小鼠。按照国家指导方针, 由调查人员和 #39 的机构动物保育和使用委员会批准, 遵循相应的动物保育和实验协议。在开始手术前至少允许7天的驯化期. </li>
	<li> 在手术前一天为所有小鼠提供镇痛, 直到第三个术后日. </li>
</ol> 2。外固定器的手术方法及应用 <ol> <li> 将鼠标置于预加载的5-7% 异氟醚和60毫升/分钟氧气管中。在失去姿势反射后, 从麻醉诱导管中取出鼠标, 并通过吸入面罩维持麻醉, 提供1-3% 异氟醚和60毫升/分钟的氧气。
	<ol> <li> 监视麻醉过程中的呼吸模式和后爪反射。确保呼吸率是大约100周期/分钟和后爪反射缺席. 
		#8203; 注意: 所需的气体量取决于年龄、性别、体重和老鼠的应变. </li>
	</ol> </li> <li> 手术前, 用单一剂量的抗生素皮下注射注射小鼠 (克林霉素, 45 毫克/千克)。此外, 为了维持生理体液平衡, 将老鼠注入500和 #181 的皮下液体库; L 生理盐水 (0.9% 氯化钠). </li>
	<li> 防止角膜干燥, 用眼膏涂抹于老鼠眼。将鼠标放在37和 #176 的加热板上; C 在麻醉和手术过程中保持生理体温. </li>
	<li> 去除右后肢的皮毛, 用酒精消毒剂擦洗手术区。用不育手套的一小部分将右后爪盖住, 以避免消毒的部位。将右后肢消毒三次。在整个鼠标上放置一个无菌的褶皱, 除了手术区. </li>
	<li> 沿右侧股骨前侧用手术刀切开大约1厘米的皮肤。用微剪刀和镊子将 m. 股二头肌 和 m. 股外 分开。切断肌腱的起源和 #160; 一侧在股骨粗隆与一个微型剪刀, 允许自由进入骨外侧部分。确保坐骨神经被保存. </li>
	<li> 将外部固定架 (轴向刚度 3/毫米, 图 1 A ) 与股骨平行。手动钻孔通过皮质与0.45 毫米钻头和安置陶瓷安装销进入钻孔。从最接近的引脚开始, 其次是最远端的引脚, 和两个引脚之间。
	<ol> <li> 在安装过程中确保固定架上没有张力、压缩或剪切应力, 否则由于固定器松弛, 达到截骨间隙将不足够. </li>
	</ol> </li> <li> 用少量的无菌氯化钠 Humidify 骨, 以避免在锯切过程中出现脱水. </li>
	<li> 通过使用 0.4 mm 吉利线锯, 在两个内针之间的整个骨骼之间创建一个 0.4 mm 截骨. 
	注: 可选地, 振荡微锯可用于创建截骨。请务必避免在截骨区的锯的任何金属芯片. </li>
	<li> 用2毫升无菌氯化钠冲洗截骨间隙, 清除两个骨折的皮质之间的骨头碎片. </li>
	<li> 通过连续缝合和可缝合来适应肌肉 (请参阅 材料表 )。然后使用间断的 non-resorbable 缝合来调整皮肤 (请参见 材料表 )。为避免伤口咬伤, 请勿将缝线放置在伤口的颅骨部位. 
	注意: 请勿使用皮肤胶或夹子, 因为小鼠通常会将其从伤口上移除, 造成皮肤进一步损伤. </li>
	<li> 用消毒剂清洗手术区, 并将鼠标放进笼子. #160; 监视鼠标并提供足够的热量 ( 如 通过红外线), 直到完全清醒为止. #160; 在手术后监测水、食物摄入量和体重确保动物没有痛苦和苦恼。通过饮用水为所有小鼠提供镇痛, 直到第三天后. 
	注: 小鼠可容纳多达四动物组. </li>
	<li> 在手术后1到5天监视鼠标和 #39 的活动。在那段时间里, 老鼠应该在操作的肢体上承受重量。否则, 必须将鼠标排除在进一步分析之外. </li>
</ol> 3。mri 检查程序和图像分析 <ol> <li> 在 mri 扫描过程之前, 根据步骤2.1 和2.3 中的协议麻醉鼠标, 并使呼吸频率保持在100周/分钟左右. 插入外部固定器在右后肢小心地将鼠标放入 custom-made 安装设备 ( 图 1 B、C )。
	<ol> <li> 确保在这一步中避免对固定架进行弯曲或压缩, 因为这可能会影响骨折愈合. 
		注: MRI 扫描可在手术后3天进行, 这取决于动物护理和实验协议. </li>
	</ol> </li> <li> 将鼠标放置在温度控制的底座上, 以便引入 MRI 设备。将安装装置刚性地连接到四元件的磁头线圈上. </li>
	<li> 使用专用的型小动物磁共振成像系统采集 mri 数据, 操作 11.7 T. 
	#8203; 注意: MRI 数据的采集几何与股骨骨正交, 并与螺钉相吻合。
	<ol> <li> 使用捕获参数通过应用质子密度脂肪抑制的多层谢志伟序列 (PD) 获取数据: 回声/重复时间 TE = 5.8 毫秒/TR = 2500 毫秒, 分辨率和 #916; r = 52 和 #215; 52 和 #215; 350 和 #181; m 和 #179;, 视 (视) =20和 #215; 20 mm 和 #178;, 带宽和 #916; #969; = 150 赫. </li>
		<li> 注意:22 切片的总获取时间为 36 min. </li>
	</ol> </li> <li> 使用图像分析软件打开获取的数据。输入体素大小为 0.05 x 0.05 x 0.35 mm 3 。在骨折愈伤组织 (骨, 软骨, 纤维组织/骨髓) 的不同组织的基础上, 其强度与半自动阈值如下。
	<ol> <li> 单击 "#34"; 编辑新标签字段和 #34; 单击和 #34; 添加材料和 #34; 并将材料重命名为和 #34; 愈伤组织和 #34;。利用 #34; 套索和 #34; 工具, 从骨膜的低强度信号中区分愈伤组织区和周围组织. </li>
		<li> 单击和 #34; 添加到材料和 #34;。单击并 #34; 添加材料和 #34; 并将材料重命名为和 #34; 软骨和 #34。使用 #34; 阈值和 #34; 工具和 #34; 选择当前的材料和 #34; 从 #34; 愈伤组织和 #34;单击和 #34; 软骨和 #34; 和 #34; 添加到材料和 #34;。重复这些步骤与 #34; 骨骼和 #34; 和 #34; 骨髓/纤维组织和 #34;... </li>
	</ol> </li> <li> 使用图像分析软件基于组织分割数据生成断裂股骨的3D 重建。单击和 #34; 生成表面和 #34; 应用和 #34; 无 #34; #34; 平滑类型和 #34; 单击和 #34; 表面视图和 #34;. 
	注: 非常小, 超强的地区周围的 en由于从骨组织到软组织的转变, 骨折皮层的 ds 可能是伪影。这些领域应排除在进一步分析之外。在骨折愈合的软骨期, 骨折愈伤组织中部的超强区域代表软骨组织。在骨折愈伤组织远端的低强度区, 在软骨骨化期的截骨间隙, 以及在整个骨折愈伤组织中, 在后期愈合阶段相同的部位, 表现为新近形成的骨痂组织。虽然这些区域有一个低强度的信号, 从成熟的骨骼 (皮质) 的信号密集度甚至较低。对骨折愈伤组织骨组织和软骨组织的信号强度进行阈值化后, 将其余组织标记为骨髓和纤维组织。组织细分的价值是: 骨组织 (包括成熟皮质, 小梁骨, 骨痂组织) 是分段范围内的 1-3. 3 (正常化信号强度到成熟皮质), 骨髓/纤维组织在 3.4-5.4 范围内, 并软骨痂组织在 5.5-6.2 的范围内. </li>
	<li> 如果需要, 在骨折愈合过程中纵向重复 MRI 扫描。为了监测软骨痂的发育, 在手术后10、14、21天扫描小鼠. 
	注意: 时间点可能取决于动物保育和实验协议. </li>
</ol>

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作者罗马 Matthys 是 RISystem AG 达沃斯的雇员, 瑞士, 生产植入物和植入在本文中使用的特定仪器。所有其他作者都没有竞争的金融利益。

修改和故障排除:
本研究的主要目的是描述一个协议的使用 MRI 兼容外固定器股骨截骨在小鼠的能力, 以监测骨痂组织发展纵向软骨骨折愈合过程。custom-made 安装装置的设计, 用于插入外固定架, 确保了在重复扫描过程中的标准化位置。半自动组织分割允许分析的数量的纤维, 软骨, 骨组织的骨折骨痂。此外, 3D 重建的 MRI 图像允许可视化的软骨骨折愈合过程中每个单?...

二次骨折愈合是最常见的骨愈合形式。这是一个复杂的过程, 模仿 ontogenic 软骨骨化的具体方面1,2,3。早期骨折血肿主要由免疫细胞、肉芽组织和纤维组织组成。低氧张力和高生物力学菌株在骨折间隙中阻碍成骨细胞分化, 但促进前体细胞分化成软骨细胞4,5,6。这些细胞在损伤部位开始增殖, 形成软骨基质, 提供骨折骨的初始稳定性。在愈伤组织成熟过程中, 软骨细胞变得肥大、凋亡或分化成成骨细胞。cartilage-to-bone 过渡区的新生血管提供了高氧水平, 使骨组织的形成7。骨折间隙骨桥接后, 生物力学稳定性提高, 外骨折愈伤组织发生破重塑, 获得生理骨轮廓和结构3。因此, 骨痂中纤维、软骨和骨组织的数量提供了骨骼愈合过程的重要信息。扰或延迟愈合变得可看见通过改变愈伤组织的发展在人和小鼠8,9,10,11。可用的体内技术纵向监测骨痂组织发育的临床前骨折愈合研究使用小动物包括数字造影和µCT 成像12,13。然而, 这两种技术只能够区分矿化和 non-mineralized 组织。相比之下, MRI 提供了优秀的软组织对比, 因此可以识别软组织和软骨在骨折愈伤组织。
先前的工作显示, 在膜骨缺损愈合15中, 在小鼠关节骨折的14和活体mri 检查中, 有希望的结果。然而, 这两项研究也表明空间分辨率和组织对比度有限。我们先前证明了高分辨率的体内 MRI 对小鼠软骨骨折愈合过程中软痂形成的纵向评估的可行性16。在这里, 我们报告的协议, 使用 MRI 兼容外固定器股骨截骨在小鼠, 以监测骨痂组织发展纵向在软骨骨折愈合过程。custom-made 安装装置的设计, 用于插入外固定架, 确保了在重复扫描过程中的标准化位置。

<table><tbody><tr><td>Anaesthesia tube</td><td>FMI, Seeheim, Germany</td><td>ZUA-82-ANA-TUB-Mouse</td><td></td></tr><tr><td>Anaesthetic machine&amp;nbsp;</td><td>FMI, Seeheim, Germany</td><td>ZUA-82-GME-MA</td><td></td></tr><tr><td>Artery forceps&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BH104R</td><td></td></tr><tr><td>Autoclave</td><td>Systec, Wettenberg, Germany</td><td>DX-150</td><td></td></tr><tr><td>Autoclaving packaging</td><td>Stericlin, Feuchtwangen, Germany</td><td>2301-04/06/10/12/16</td><td></td></tr><tr><td>Avizo software</td><td>FEI, Burlington, USA</td><td>-</td><td>Version 8.0.1</td></tr><tr><td>BioSpec 117/16 magnetic resonance imaging system</td><td>Bruker Biospin, Ettlingen, Germany</td><td>117/16</td><td></td></tr><tr><td>Bulldog clamp&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BH 021R</td><td></td></tr><tr><td>Carbon steel scalpel no. 11/15</td><td>Aesculap, Tuttlingen, Germany</td><td>BA211/215</td><td></td></tr><tr><td>Ceramic mounting pin 0.45 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>HS691490</td><td></td></tr><tr><td>Clindamycin (300 mg / 2ml)</td><td>Ratiopharm, Ulm, Germany</td><td>-</td><td></td></tr><tr><td>Dressing forceps 115 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD210R</td><td></td></tr><tr><td>Dressing forceps 130 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD025R</td><td></td></tr><tr><td>Drill bit coated 0.45 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>HS820420</td><td></td></tr><tr><td>Durogrip needle holder 125 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BM024R</td><td></td></tr><tr><td>Foliodrape&amp;nbsp;</td><td>Hartmann, Heidenheim, Germany</td><td>2513026</td><td></td></tr><tr><td>Frekaderm</td><td>Fresenius, Bad Homburg, Germany</td><td>4928211</td><td></td></tr><tr><td>Gigli saw 0.44 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>RIS.590.110.25</td><td></td></tr><tr><td>Hand drill</td><td>RISystem, Davos, Switzerland</td><td>RIS.390.130-01</td><td></td></tr><tr><td>Heating plate&amp;nbsp;</td><td>FMI, Seeheim, Germany</td><td>IOW-3704</td><td></td></tr><tr><td>Hygonorm gloves&amp;nbsp;</td><td>Hygi, Telgte, Germany</td><td>2706</td><td></td></tr><tr><td>Isoflurane</td><td>Abbot, London, UK</td><td>Forene</td><td></td></tr><tr><td>Micro forceps 155 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BD343R</td><td></td></tr><tr><td>Micro scissors 120 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>FD013R</td><td></td></tr><tr><td>Mouse FixEx L 0.7 mm&amp;nbsp;</td><td>RISystem, Davos, Switzerland</td><td>RIS.611.300-10</td><td></td></tr><tr><td>Needle case for drills&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BL911R</td><td></td></tr><tr><td>Needle holder</td><td>Aesculap, Tuttlingen, Germany</td><td>BB078R</td><td></td></tr><tr><td>Octenisept</td><td>Sch&amp;uuml;lke, Norderstedt, Germany</td><td>121403</td><td></td></tr><tr><td>Osirix software</td><td>Pixmeo SARL, Bernex, Switzerland</td><td>-</td><td>Version 4.0</td></tr><tr><td>Oxygen, medical grade</td><td>MTI, Ulm, Germany</td><td>-</td><td></td></tr><tr><td>Resolon 5/0</td><td>Resorba, N&amp;uuml;rnberg, Germany</td><td>88143</td><td></td></tr><tr><td>Saline 0.9%</td><td>Braun, Melsungen, Germany</td><td>3570350</td><td></td></tr><tr><td>Scalpel handle 125 mm</td><td>Aesculap, Tuttlingen, Germany</td><td>BB073R</td><td></td></tr><tr><td>Scissors 150 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BC006R</td><td></td></tr><tr><td>Sealer for autoclave packaging&amp;nbsp;</td><td>Hawo GmbH, Obrigheim, Germany</td><td>HM500</td><td></td></tr><tr><td>Sterican 27 G&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>4657705</td><td></td></tr><tr><td>Sterile surgical blades no. 11/15&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>BB511/515</td><td></td></tr><tr><td>Surgical gloves&amp;nbsp;</td><td>Hartmann, Heidenheim, Germany</td><td>Peha-micron 9425712</td><td></td></tr><tr><td>Surgical light&amp;nbsp;</td><td>Maquet SA, Ardon, France</td><td>Blue line 80</td><td></td></tr><tr><td>Syringes 5 ml&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>Injekt 4606051V</td><td></td></tr><tr><td>Tissue forceps 80 mm&amp;nbsp;</td><td>Aesculap, Tuttlingen, Germany</td><td>OC091R</td><td></td></tr><tr><td>Tramadol 25 mg/l</td><td>Gr&amp;uuml;nenthal, Aachen, Germany</td><td>100mg/ml</td><td></td></tr><tr><td>Vasofix Safety&amp;nbsp;</td><td>Braun, Melsungen, Germany</td><td>4268113S-01</td><td></td></tr><tr><td>Vicryl 5-0&amp;nbsp;</td><td>Ethicon, Norderstedt, Germany</td><td>V30371</td><td></td></tr><tr><td>Visdisic eye ointment&amp;nbsp;</td><td>Bausch &amp;amp; Lomb, Berlin, Germany</td><td>3099559</td><td></td></tr></tbody></table>

in vivo evaluation of fracture callus development during bone healing in mice using an mri-compatible osteosynthesis device for the mouse femur

软骨骨折愈合是一个复杂的过程, 涉及发展的纤维, 软骨, 骨组织在骨折愈伤组织。骨痂中不同组织的数量为骨折愈合的进展提供了重要的信息。可用的体内技术纵向监测骨痂组织的发展在临床前骨折愈合研究中使用小动物包括数字造影和µCT 成像。然而, 这两种技术只能够区分矿化和 non-mineralized 组织。因此, 不可能区分软骨和纤维组织。相比之下, 磁共振成像 (MRI) 可视的解剖结构的基础上, 其含水量, 从而可以非识别软组织和软骨的骨折愈伤组织。在这里, 我们报告了使用 mri 兼容外固定器的小鼠股骨, 允许 mri 扫描在小鼠骨再生。实验表明, 该固定架和 custom-made 安装装置允许重复 MRI 扫描, 从而使纵向分析骨折-愈伤组织的发展。

首先, 通过对 MRI 扫描的分析可以确认手术的成功 (参见图 2中的示例)。所有四针应位于股骨干中间。截骨间隙的大小应介于 0.3-0.5 毫米之间。如果截骨间隙的大小与这些值相差很大, 则应将鼠标排除在进一步分析之外。
其次, 对同一动物骨折愈合过程中纵向扫描的评价提供了有关愈伤组织发育的信息。如?...

Watch this Scientific Journal Video about In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur at JoVE.com

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

在体内MRI 对小鼠股骨骨愈合过程中骨折愈伤组织发育的评价

软骨骨愈合过程中骨折愈伤组织发育的评价是监测愈合进程的关键。在这里, 我们报告的使用磁共振成像 (mri) 兼容外固定器的小鼠股骨, 允许 mri 扫描在小鼠骨再生。

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

in-vivo-evaluation-fracture-callus-development-during-bone-healing

Research

JoVE Journal

Medicine

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

9.5K 次观看.  University Medical Center Ulm. 软骨骨愈合过程中骨折愈伤组织发育的评价是监测愈合进程的关键。在这里, 我们报告的使用磁共振成像 (mri) 兼容外固定器的小鼠股骨, 允许 mri 扫描在小鼠骨再生。

视频: In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

科研

医学

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

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

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

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

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

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

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

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

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

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

髓内锁钉股骨截骨标准化固定用于分析正常和有缺陷的骨愈合小鼠

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

A Minimally Invasive Model to Analyze Endochondral Fracture Healing in Mice Under Standardized Biomechanical Conditions

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an increased use of mouse models in orthopedic research was noted, most probably because mouse models offer a large number of genetically-modified strains and special antibodies for the analysis of molecular mechanisms of fracture healing. To control the biomechanical conditions, well-characterized osteosynthesis techniques are mandatory, also in mice. Here, we report on the design and use of a closed bone healing model to stabilize femur fractures in mice. The intramedullary screw, made of medical-grade stainless steel, provides through fracture compression an axial and rotational stability compared to the mostly used simple intramedullary pins, which show a complete lack of axial and rotational stability. The stability achieved by the intramedullary screw allows the analysis of endochondral healing. A large amount of callus tissue, received after stabilization with the screw, offers ideal conditions to harvest tissue for biochemical and molecular analyses. A further advantage of the use of the screw is the fact that the screw can be inserted into the femur with a minimally invasive technique without inducing damage to the soft tissue. In conclusion, the screw is a unique implant that can ideally be used in closed fracture healing models offering standardized biomechanical conditions.

This protocol describes a minimally invasive osteosynthesis technique using an intramedullary screw for standardized stabilization of femur fractures, which can be used to analyze endochondral bone healing in mice.

标准化生物力学条件下小鼠软骨骨折愈合的微创模型分析

Bone healing models are necessary to analyze the complex mechanisms of fracture healing to improve clinical fracture treatment. During the last decade, an ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Protocol for Developing a Femur Osteotomy Model in Wistar Albino Rats

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

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

开发Wistar白化大鼠股骨截骨模型的协议

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

Transverse Fracture of the Mouse Femur with Stabilizing Pin

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test whether a gene, gene product or drug influences bone repair because murine bones recapitulate the stages observed during human fracture healing. When a mouse or human breaks a bone, an inflammatory response is initiated, and the periosteum, a stem cell niche surrounding the bone itself, is activated and expands. Cells residing in the periosteum then differentiate to form a vascularized soft callus. The transition from the soft callus to a hard callus occurs as the recruited skeletal progenitor cells differentiate into mineralizing cells, and the bridging of the fractured ends results in the bone union. The mineralized callus then undergoes remodeling to restore the original shape and structure of the healed bone. Fracture healing has been studied in mice using various injury models. Still, the best way to recapitulate this entire biological process is to break through the cross-section of a long bone that encompasses both cortices. This protocol describes how a stabilized, transverse femur fracture can be safely performed to assess healing in adult mice. A surgical protocol including detailed harvesting and imaging techniques to characterize the different stages of fracture healing is also provided.

This protocol describes a method to perform fractures on adult mice and monitor the healing process.

带稳定针的小鼠股骨横断

Fracture repair is an essential function of the skeleton that cannot be reliably modeled in vitro. A mouse injury model is an efficient approach to test ...

生物学

Assessment of Bone Fracture Healing Using Micro-Computed Tomography

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed bone during fracture healing in translational science investigations. Studies of long bone fracture healing in rodents typically involve secondary healing and the formation of a mineralized callus. The shape of the callus formed and the density of the newly formed bone may vary substantially between timepoints and treatments. Whereas standard methodologies for quantifying parameters of intact cortical and trabecular bone are widely used and embedded in commercially available software, there is a lack of consensus on procedures for analyzing the healing callus. The purpose of this work is to describe a standardized protocol that quantitates bone volume fraction and callus mineral density in the healing callus. The protocol describes different parameters that should be considered during imaging and analysis, including sample alignment during imaging, the size of the volume of interest, and the number of slices that are contoured to define the callus.

Micro-computed tomography (&#181;CT) is a non-destructive imaging tool that is instrumental in assessing bone structure in preclinical studies, however there is a lack of consensus on &#181;CT procedures for analyzing the bone healing callus. This study provides a step-by-step &#181;CT protocol that allows the monitoring of fracture healing.

使用显微计算机断层扫描评估骨折愈合

Micro-computed tomography (&#181;CT) is the most common imaging modality to characterize the three-dimensional (3D) morphology of bone and newly formed ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

建立小鼠骨干股骨骨折模型

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...