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独的鼠标。
协议中的关键步骤:
手术过程中最关键的步骤是使用 MRI 兼容的外固定支架: (1) 在手术中避免坐骨神经损伤, 否则, 在截骨术后5天内, 小鼠就不能负重, 必须排除进一步分析。(2) 在安装过程中, 避免固定架上的张力、压迫或剪切应力, 否则截骨间隙不会有一个标准化的尺寸和形状。此外, 一定要安装与股骨纵轴平行的固定架, 确保截骨的稳定固定。(3) 避免金属芯片从锯, 如果使用吉利线锯, 因为那些将干扰核磁共振扫描程序。
MRI 扫描过程中最关键的步骤是: (1) 在安装装置的插入和拆卸过程中, 一定要避免固定架的弯曲或压迫, 因为这可能会影响骨折愈合。(2) 在扫描过程中确保适当的温度控制, 以保持生理体温。
在现有方法和技术限制方面的重要性:
以前的研究显示有希望的结果宰后磁共振成像在小鼠关节骨折14和体内mri 在小鼠膜骨缺损愈合15。然而, 这两项研究也表明空间分辨率和组织对比度有限。我们以前展示了高分辨率的在体内mri 对小鼠骨折愈合早期和中期的软愈伤组织形成进行纵向分析的可行性和准确性, 并将新的 mri 技术与金标准µCT 和计量16。然而, 我们也发现, MRI 的空间分辨率明显低于前体µCT 的分辨率。这是一个明确的局限性, MRI 技术时相比, 竞争技术, 包括ex 体内, 但也在体内µCT。
未来应用:
磁共振成像在小鼠骨折愈合研究中的应用前景是: (1) mri 与造影剂结合使用, 测量血流通过损伤肢体。(2) MRI 与 PET 扫描相结合, 以及用顺颗粒标记细胞进行细胞贩运实验17,18,19,20。

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。

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			<td class="xl65" height="21" style="width:254pt;height:15.75pt" width="338">Anaesthesia tube</td>
			<td class="xl65" style="width:295pt" width="393">FMI, Seeheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">ZUA-82-ANA-TUB-Mouse</td>
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			<td class="xl65" height="21" style="height:15.75pt">Anaesthetic machine&#160;</td>
			<td class="xl65">FMI, Seeheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">ZUA-82-GME-MA</td>
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			<td class="xl65" height="21" style="height:15.75pt">Artery forceps&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BH104R</td>
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			<td class="xl65" height="21" style="height:15.75pt">Autoclave</td>
			<td class="xl65">Systec, Wettenberg, Germany</td>
			<td class="xl64" style="width:149pt" width="199">DX-150</td>
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			<td class="xl65" height="21" style="height:15.75pt">Autoclaving packaging</td>
			<td class="xl65">Stericlin, Feuchtwangen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">2301-04/06/10/12/16</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Avizo software</td>
			<td class="xl65">FEI, Burlington, USA</td>
			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67">Version 8.0.1</td>
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			<td class="xl65" height="21" style="height:15.75pt">BioSpec 117/16 magnetic resonance imaging system</td>
			<td class="xl65">Bruker Biospin, Ettlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">117/16</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Bulldog clamp&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BH 021R</td>
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			<td class="xl65" height="21" style="height:15.75pt">Carbon steel scalpel no. 11/15</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BA211/215</td>
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			<td class="xl65" height="21" style="height:15.75pt">Ceramic mounting pin 0.45 mm&#160;</td>
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			<td class="xl64" style="width:149pt" width="199">HS691490</td>
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			<td class="xl65" height="21" style="height:15.75pt">Clindamycin (300 mg / 2ml)</td>
			<td class="xl65">Ratiopharm, Ulm, Germany</td>
			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Dressing forceps 115 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BD210R</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Dressing forceps 130 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BD025R</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Drill bit coated 0.45 mm&#160;</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">HS820420</td>
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			<td class="xl65" height="21" style="height:15.75pt">Durogrip needle holder 125 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BM024R</td>
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			<td class="xl65" height="21" style="height:15.75pt">Foliodrape&#160;</td>
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			<td class="xl64" style="width:149pt" width="199">2513026</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Frekaderm</td>
			<td class="xl65">Fresenius, Bad Homburg, Germany</td>
			<td class="xl64" style="width:149pt" width="199">4928211</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Gigli saw 0.44 mm&#160;</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">RIS.590.110.25</td>
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			<td class="xl65" height="21" style="height:15.75pt">Hand drill</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">RIS.390.130-01</td>
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			<td class="xl65" height="21" style="height:15.75pt">Heating plate&#160;</td>
			<td class="xl65">FMI, Seeheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">IOW-3704</td>
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			<td class="xl65" height="21" style="height:15.75pt">Hygonorm gloves&#160;</td>
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			<td class="xl64" style="width:149pt" width="199">2706</td>
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			<td class="xl65" height="21" style="height:15.75pt">Isoflurane</td>
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			<td class="xl64" style="width:149pt" width="199">Forene</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Micro forceps 155 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BD343R</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Micro scissors 120 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">FD013R</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Mouse FixEx L 0.7 mm&#160;</td>
			<td class="xl65">RISystem, Davos, Switzerland</td>
			<td class="xl64" style="width:149pt" width="199">RIS.611.300-10</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Needle case for drills&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BL911R</td>
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			<td class="xl65" height="21" style="height:15.75pt">Needle holder</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BB078R</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Octenisept</td>
			<td class="xl65">Sch&#252;lke, Norderstedt, Germany</td>
			<td class="xl64" style="width:149pt" width="199">121403</td>
			<td class="xl67"></td>
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			<td class="xl65" height="21" style="height:15.75pt">Osirix software</td>
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			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67">Version 4.0</td>
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			<td class="xl65">MTI, Ulm, Germany</td>
			<td class="xl64" style="width:149pt" width="199">-</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Resolon 5/0</td>
			<td class="xl65">Resorba, N&#252;rnberg, Germany</td>
			<td class="xl64" style="width:149pt" width="199">88143</td>
			<td class="xl67"></td>
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			<td class="xl65">Braun, Melsungen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">3570350</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Scalpel handle 125 mm</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BB073R</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Scissors 150 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BC006R</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Sealer for autoclave packaging&#160;</td>
			<td class="xl65">Hawo GmbH, Obrigheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">HM500</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Sterican 27 G&#160;</td>
			<td class="xl65">Braun, Melsungen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">4657705</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Sterile surgical blades no. 11/15&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">BB511/515</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Surgical gloves&#160;</td>
			<td class="xl65">Hartmann, Heidenheim, Germany</td>
			<td class="xl64" style="width:149pt" width="199">Peha-micron 9425712</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Surgical light&#160;</td>
			<td class="xl65">Maquet SA, Ardon, France</td>
			<td class="xl64" style="width:149pt" width="199">Blue line 80</td>
			<td class="xl67"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Syringes 5 ml&#160;</td>
			<td class="xl65">Braun, Melsungen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">Injekt 4606051V</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Tissue forceps 80 mm&#160;</td>
			<td class="xl65">Aesculap, Tuttlingen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">OC091R</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Tramadol 25 mg/l</td>
			<td class="xl65">Gr&#252;nenthal, Aachen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">100mg/ml</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Vasofix Safety&#160;</td>
			<td class="xl65">Braun, Melsungen, Germany</td>
			<td class="xl64" style="width:149pt" width="199">4268113S-01</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Vicryl 5-0&#160;</td>
			<td class="xl65">Ethicon, Norderstedt, Germany</td>
			<td class="xl64" style="width:149pt" width="199">V30371</td>
			<td class="xl67"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="height:15.75pt">Visdisic eye ointment&#160;</td>
			<td class="xl65">Bausch &#38; Lomb, Berlin, Germany</td>
			<td class="xl64" style="width:149pt" width="199">3099559</td>
			<td class="xl67"></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。如果在10、14和21天扫描鼠标 (参见图 3中的示例), 软骨组织在每天 10 (相对软骨面积 = 30.8%) 和天 14 (相对软骨面积 = 29.0%) 的骨折愈伤组织的中部可见, 并减少到21天后手术 (相对软骨面积 = 10.5%)(图 3)。骨组织是可看见的在断裂愈伤组织的边缘在天 10 (相对骨头区域 = 7.2%), 增加直到天 14 (相对骨头区域 = 15.6%) 和身体桥梁发生直到天 21 (相对骨头区域 = 45.7%)。
第三, 利用图像分析软件对骨折愈伤组织的不同组织进行分割后, 可产生股骨骨折和骨痂的3D 图像。在图 4中所示的示例中, 显示了在断开后26天扫描的整个股骨。成熟皮质标记为灰色, 陶瓷针标记为黄色, 愈伤组织软组织标记为绿色, 软骨组织呈红色标记, 骨痂组织呈紫色。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56679/56679fig1.jpg"> 
图 1: 带有陶瓷安装销和 MRI 安装装置的外固定器.(A) 显示外部固定架的塑料体, 以及与 MRI 扫描相兼容的四陶瓷安装销。缩放栏: 1 厘米 (B) 在 MRI 扫描中, custom-made 安装装置用于插入外固定架的计算机辅助绘图显示。将鼠标右股骨的外固定器插入安装装置的浮雕中。然后, 在扫描前将该设备插入四元磁头线圈上。缩放栏: 0.4 厘米 (C) 鼠标放置在安装设备 (蓝色), 连接到4元头线圈 (白色)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56679/56679fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56679/56679fig2.jpg"> 
图 2: 手术后3天股骨骨折的 MRI 影像.手术后3天扫描的大腿骨折的中央切片显示。骨髓;B: 骨头;FX: 骨折间隙。缩放栏: 0.5 mm<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56679/56679fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56679/56679fig3.jpg"> 
图 3: 利用 MRI 技术对骨折愈伤组织发育进行纵向监测.中央 MRI 切片从骨折一只鼠标扫描 (A) 天 10, (B) 天 14, 和 (C) 天21后手术显示。超强的软骨组织在10天和14日的骨折愈伤组织中部可见, 术后21天减少。10天, 在骨折愈伤组织的周围可见低强度的骨组织, 增加至14天, 身体架桥发生到21天。骨髓;软骨组织;骨组织。缩放栏: 0.5 mm<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56679/56679fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56679/56679fig4.jpg"> 
图 4: 手术后26天扫描的股骨骨折的3D 重建.成熟皮质标记为灰色, 陶瓷针标记为黄色, 愈伤组织软组织标记为绿色, 软骨组织呈红色标记, 骨痂组织呈紫色。图像是使用图像分析软件生成的。缩放栏: 0.4 mm<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56679/56679fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

鼠标使用新型脊髓损伤的后肢骨质流失的纵向评价，在体内，方法论

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

科研

医学

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

基于音频的虚拟游戏环境的发展，以帮助盲人的导航技巧

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

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 ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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 ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

伤口的牙髓评价一个直接盖髓模型的开发和治疗小鼠中修复性牙本质形成

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

用于评估椎弓根螺钉的麻醉，手术和收获方法<em&gt;体内</em&gt;猪腰椎模型

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

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 ...