作者感谢罗切斯特大学肌肉骨骼研究中心组织学、生物化学和分子成像 （HBMI） 核心的 Jeff Fox 和 Vidya Venkatramani 提供的支持和帮助，部分资金来自 P30AR06965。此外，作者要感谢罗切斯特大学医学中心的光学显微镜和纳米镜中心（CALMN）在多光子显微镜方面的帮助。这项研究由 R01 AR070765 和 R01 AR070765-04S1 以及 1R35GM147054 和 1R01AR082349 资助。

所有动物工作均由罗切斯特大学动物资源委员会批准。
1.组织培养基的制备
<ol><li>在37°C和5%CO2的培养箱中，在Dulbecco改良的Eagle培养基（1x DMEM）中培养所有外植体，其中含有1%v / v青霉素 - 链霉素和200μM L-抗坏血酸。对于最初的 48 小时预处理，在补充有 100 nM 地塞米松74 的 70 mL 培养基中培养每个外植体。预处理后，在没有地塞米松的情况下再培养肢体7天，每48-72小时更换一次培养基。 注：不建议将血清（如胎牛血清）添加到培养基中，这与 Wunderli、Blache 和 Snedeker 提供的建议一致57。简而言之，无血清条件更能代表 体内存在的无血管、营养不良的肌腱微环境。此外，在某些培养条件下，血清补充剂可以促进组织降解75 ，并刺激细胞增殖和从组织中迁移，这两者都是肌腱病理学的特征57。</li>
	<li>对于未上样的组，在 70 mL 培养基中培养每个外植体。对于基线张力组和静态撞击组，每个平台需要大约 125 mL 的培养基来保持肢体浸没。该体积可能因大腿在握把中的位置和 3D 打印参数（主要是填充密度）而异。</li>
</ol>2.外植体解剖和地塞米松预处理
<ol><li>通过吸入 CO2 和继发性颈椎脱位或根据机构指南对小鼠实施安乐死。该方案利用小于1岁的C57BL / 6小鼠。后肢的大小会随着年龄的增长而增加，并且可能变得难以适应握把。</li>
	<li>在解剖之前，将70mL预热（37°C）培养基转移到无菌生物安全柜（BSC）中的100mm（直径）x 25mm（高度）培养皿中。加入地塞米松以达到 100 nM 工作浓度。</li>
	<li>解剖可以使用吸收性底垫在工作台上进行，在将后肢外植体转移到 BSC 之前迅速完成解剖。组装解剖所需的手术工具，包括光滑、笔直、细尖镊子;笔直、细尖的镊子，带有锯齿状;和直的、锋利的、细的剪刀。</li>
	<li>对于后肢外植体的解剖，将小鼠置于仰卧位并识别髋关节。使用细剪刀，在大腿近端和前部（颅面）的皮肤上做一个小切口（5-10 毫米）。</li>
	<li>将切口拉开以扩大，用手指捏住裸露的大腿，然后小心地将皮肤向远端拉动，使后肢脱屑至脚踝水平。沿着脚背轻轻地将一把剪刀刀片插入皮肤下，并切开一个延伸到脚趾的切口。继续向远端拉扯皮肤以完全去除。</li>
	<li>放置鼠标以可视化跟腱插入跟骨的后（尾）侧，靠近脚踝。足底肌腱位于跟腱插入的近端，紧邻跟腱的内侧缘，向足底远端延伸，越过跟骨后侧。</li>
	<li>要去除足底肌腱，请小心地将光滑、细尖钳的尖端插入两个肌腱之间，并将尖端向内侧延伸，穿过足底肌腱下方。将尖端向近端拉出并撕裂足底肌肉。使用细尖锯齿状镊子，抓住足底肌腱分离的近端并向远端拉动以将其移除。</li>
	<li>在髋关节处，用细剪刀切开骨盆，隔离后肢。用剪刀撬开剩余的骨盆，露出股骨头。</li>
	<li>将后肢外植体转移到 BSC 中，并转移到含有地塞米松的细胞培养基的培养皿中。将培养皿移入培养箱并预处理48小时。</li>
</ol>3. 外植体培养和装载平台
<ol><li>随着48小时预处理的结束，预热足够体积的培养基（第1节）。从现在开始，地塞米松不会添加到培养基中。此时，可以将预处理组（第 0 天）的肢体固定、脱钙并包埋在石蜡中，以便将来进行切片、染色和分析。</li>
	<li>对于未加载的组，吸出预处理培养基并将外植体转移到新鲜的培养皿中，每个加入 70 mL 培养基，然后返回培养箱。</li>
	<li>对于基线张力组和静态冲击组，准备外植体平台。切割大小与握把压板相似的砂纸片。对于静电冲击组，将编织线剪成约 18 英寸长的碎片，并沿线长度的一半预打一个松散的反手结。撕下铝箔片以覆盖每个丙烯酸浴，并喷洒 70% 乙醇 （EtOH）。将准备工作转移到平衡计分卡。</li>
	<li>每个平台都包括一个亚克力浴缸、底座、减容插件、夹子和把手。每个夹具包括两个压板和三种不同类型的螺钉，包括一个 M5 x 0.8 mm 螺纹 x 10 mm 长的螺钉，用于将夹具连接到底座;两个 M6 x 1 mm 螺纹 x 20 mm 长的螺钉，可延伸压板以夹紧夹具;以及四个 M3 x 0.5 mm 螺纹 x 14 mm 长的螺钉，这些螺钉与四个压缩弹簧相结合，可缩回压板以打开夹具。</li>
	<li>将所有组分放入能够在发生泄漏时捕获所有培养基的二级容器中。浸入≥10%漂白剂溶液中并浸泡至少1小时。用自来水冲洗漂白剂溶液（根据需要使用高压灭菌器）并进入 BSC。 注意： 为了排除污染，请考虑对所有自来水进行高压灭菌或使用纯净水。有关解决污染问题的其他提示，请参阅 讨论 。</li>
	<li>使用 M3 螺钉和压缩弹簧将压板连接到夹具上，使用 M5 螺钉将夹具固定到底座上，然后插入 M6 螺钉，直到它们与压板啮合。使用双面胶带将砂纸粘在压板上，然后关闭把手以促进砂纸与压板的粘合。对所有平台重复上述步骤。</li>
	<li>当准备加载平台时，完全打开把手并使用镊子，将大腿和膝盖放在压板之间，跟腱的表面朝上（图1A）。松散地关闭把手以轻轻固定到位。</li>
	<li>使用镊子抓住暴露的股骨头或股骨头，并操纵膝关节屈曲角度，同时逐渐合上把手以固定到位。对于基线张力组，如前所述伸展膝关节。当握把随着膝盖伸展而收紧时，脚踝应该自然伸展。对于静态撞击组，如前所述，在握把之间弯曲膝关节。</li>
	<li>对于静态撞击组，将绳子的反手结放在远端爪子周围并收紧。将绳子穿过位于外植体下方的底座上的槽并穿过夹孔。将底座放入亚克力浴中，并将夹子固定在浴槽的顶部边缘（图1A）。</li>
	<li>拉动绳子，使脚相对于胫骨至少 110°，并在绳子离开夹子时使用永久性记号笔标记绳子。在这个位置拍摄外植体的照片，以量化背屈角（图1A）。</li>
	<li>取下底座并沿底座上的轨道滑动以连接减容插件。将底座（现在连接到减容插件）放回亚克力浴中，并将夹子重新定位在顶部边缘。以标记的绳子为引导，拉动绳子以返回原来的背屈角度，并用胶带将绳子固定在浴缸的外面，以保持静态的背屈。</li>
	<li>对于基线张力组，只需将外植体定位在握把之间后，将带有体积减小插件的底座放入亚克力浴中，并拍照以量化背屈角。</li>
	<li>向每个平台中加入 125 mL 预热 （37 °C） 培养基以浸没外植体。用铝箔盖住浴槽的顶部，放入辅助容器中，然后进入培养箱。培养7天，每48-72小时更换培养基。 注意： 在浴缸顶部放置胶带可以防止 PLA 部件漂浮。</li>
</ol>4.固定、脱钙和石蜡包埋
<ol><li>外植体培养后，用剪刀修剪脚趾甲/远端脚趾，并切掉跟腱肌腱交界处近端的大腿。将每个修剪过的踝关节放入衬有泡沫活检垫的加工盒中。将脚踝推入盒角，使脚踝处于大约 90° 的背屈位置，然后关闭盒以保持到位。</li>
	<li>在 10% 中性缓冲福尔马林 （NBF） 中固定 3 天，并在溶解在蒸馏水 （diH2O） 中的 14% 乙二苯四乙酸 （EDTA） 中脱钙 2 周，pH 值用冰醋酸调节至 7.4-7.6。</li>
	<li>为了去除盐分，在 1x 磷酸盐缓冲盐水 （PBS） 中彻底冲洗样品 3 次，然后用 diH2O 彻底冲洗，每次 5 分钟。对石蜡组织学进行常规样品处理：通过一系列分级的 EtOH 脱水，在二甲苯中清除，然后用石蜡浸润。将样品定向并嵌入石蜡中，以通过跟腱获得矢状组织切片，如下文第5节所述（图1B）。</li>
</ol>5. 组织切片
<ol><li>使用切片机，小心地修剪到样品中，直到切片平行于块面。从关节的内侧粗略地修剪到脚踝，在到达跟腱插入的内侧边界之前停止。</li>
	<li>将样品转移到冰块中以调节温度和水合作用，并更换刀片（或转移到当前刀片的新鲜部分）。继续以 10 μm 厚度切片样品，并用明场显微镜仔细识别跟腱插入的入口。一旦确定，通过整个跟腱插入进行连续切片跟踪切片编号（即组织深度）。</li>
</ol>6. 脱蜡/补水和玻片选择
<ol><li>对于下面的每个测定，从每对对侧肢体中选择水平匹配的组织切片。染色前，放在载玻片架上，经过 3 次二甲苯更换、2 次更换 100% EtOH、2 次更换 95% EtOH 和 1 次更换 70% EtOH，每次 5 分钟。在 diH2O 中完成补水。</li>
</ol>7. TUNEL 评估跟腱活力
<ol><li>对于TUNEL标签，请根据制造商的方案进行染色。在室温下在20μg/ mL蛋白酶K中孵育20分钟，并在diH2O中冲洗。 在diH2O.中冲洗，用含有DAPI和盖玻片的抗淬灭试剂安装。</li>
	<li>使用带有 4 倍物镜的荧光显微镜对跟腱插入进行成像。包括一个DAPI通道（激发/发射波长= 360/460 nm）以可视化所有细胞核，一个TUNEL（TMR红色）通道（激发/发射波长= 540/580 nm）以可视化凋亡细胞核，如果可能的话，还包括一个明场通道。</li>
	<li>对于图像分析，将图像导入适用于基于 ROI 的处理的图像分析软件，例如 FIJI/ImageJ 或 MATLAB。定义一个感兴趣的区域（ROI），勾勒出整个跟腱（图2A），排除表腱中的细胞，这些细胞在被放入培养物中时极易因夹层和环境条件的突然变化而死亡。</li>
	<li>为此，请在 MATLAB 中通过导入明场图像并使用 drawpolygon（） 函数跟踪和包围肌腱的边界来执行 ROI 选择。MATLAB 为 ROI 创建一个 Polygon 对象，然后可以将其应用于 DAPI 和 TUNEL 通道图像，以使用 createMask（） 屏蔽 ROI 之外的像素强度数据，以便仅分析跟腱内的细胞核。</li>
	<li>导入 DAPI 和 TUNEL 通道图像，并通过归一化到非活组织（如骨骼、肌肉或脂肪）中凋亡核的最大荧光强度来确定用于定义凋亡 （TUNEL+） 细胞核的荧光强度阈值，这些组织在组织切片中偶然捕获。图像被遮蔽后，计算跟腱内凋亡核（TUNEL+核/DAPI核）的比例。</li>
</ol>8. 甲苯胺蓝组织学表征纤维软骨形成
<ol><li>一旦再水化（第 6 节），将具有水平匹配组织切片的载玻片架从对侧外植体对转移到 0.4% w/v 甲苯胺蓝 O 的 0.1 M 醋酸钠缓冲液中，使用 glacial 乙酸调节 pH 至 4.0。在室温下孵育10分钟，然后在diH2O中冲洗3次，每次30秒。</li>
	<li>通过三次更换 95% EtOH 和两次更换 100% EtOH 脱水，每次 30 秒。通过三次更换二甲苯清除，每次 1 分钟。盖玻片与二甲苯基封固剂。</li>
	<li>获取跟腱插入的 24 位红蓝绿 （RGB） 彩色图像。例如，对于此协议，使用适配器将数码彩色相机连接到具有 4 倍物镜的简单明场显微镜的目镜。</li>
	<li>为了量化跟腱插入处受压肌腱纤维软骨（CTF）16内甲苯胺蓝染色的差异（图3A，B），将RGB图像导入能够定义和管理多个ROI的图像分析软件中。选项包括 FIJI/ImageJ 中的选择和 ROI 管理器工具或 MATLAB 中的图像处理工具箱。首先设置像素/长度比例。 注意：软件选择由研究人员自行决定，当然不限于 MATLAB 或 FIJI/ImageJ。作者提供了 MATLAB 代码（补充文件 2）、描述实现的文档（补充文件 3）和示例图像（补充文件 4）。我们鼓励研究人员根据需要或首选将此代码翻译成其他编程语言，以便在其他软件中使用。</li>
	<li>通过所选软件中显示的图像，确定深肌腱边界与跟骨的交点。从这里开始，沿着深肌腱边界近端追踪 800 μm，以建立 CTF 的深边界。例如，使用 MATLAB 中的 drawpolyline（） 函数以交互方式在 RGB 图像上绘制多段线。MATLAB 创建一个包含线顶点的折线对象，该对象可以处理并修剪为 800 μm 的长度。</li>
	<li>从该位置，通过绘制一条连接到垂直于局部纤维方向的浅表肌腱边界的线段来创建 CTF 的近端边界。</li>
	<li>回到深肌腱边界和跟骨的交点，并通过沿着将 CTF 与附着区纤维软骨 （AZF） 分开的明显潮标绘制一条连接到浅表肌腱边界的线段来定义 CTF 的远端边界16（图 3A，B）。最后，通过追踪要封闭的浅表肌腱边界来生成 CTF 的浅表边界。</li>
	<li>为了描述插入处GAG染色的空间变化，将总CTF分为4个象限（图3A，B）。将深部和浅表 CTF 边界的中点与线段连接起来，以创建远端/近端边界。穿过该边界的中点，沿纤维方向连接远端和近端 CTF 边界的中点，以创建浅层/深层边界。</li>
	<li>这 6 个边界提供了可用于定义代表整个 CTF 的 ROI 的信息，以及细分 CTF 的 4 个象限。例如，在 MATLAB 中，将定义每个单独 ROI（CTF，象限 1-4）边界的顶点编译为向量，并使用 images.roi.Polygon（） 为每个 ROI 生成封闭的 Polygon 对象。</li>
	<li>定义 ROI 后，使用 FIJI 中的颜色转换器插件、MATLAB 中的 rgb2hsv（） 函数或其他应用适当变换方程的软件将 RGB 像素数据转换为色相饱和值 （HSV） 颜色空间76。然后，HSV数据可以投射到2D色相饱和空间中，其中色相和饱和度的每个组合都编码了一种独特的颜色（图3C）。</li>
	<li>在每个 ROI 中，计算描述 ROI 中平均染色颜色的平均色调和饱和度。例如，这可以在 MATLAB 中实现，通过使用定义每个 ROI 的 Polygon 对象来使用 createMask（） 遮罩图像，以便专门分析每个 ROI 中的色调饱和度像素数据。</li>
	<li>在骨膜纤维软骨（PF）16内定义另一个小的ROI（图3A，B）并计算平均色调和饱和度。然后，计算将 CTF 的每个 ROI 中的平均颜色与 PF 的平均颜色分开的欧几里得距离（图 3C）。 <img alt="Equation 1" src="/files/ftp_upload/65801/65801eq01v2.jpg" style="margin: auto;"> 注意：欧几里得距离计算描述了 ROI 的平均颜色与 PF（一种典型的纤维软骨组织 16,17,77）的平均颜色之间的相似程度。较小的欧几里得距离表示外观上更像纤维软骨的 ROI 颜色。</li>
	<li>使用此距离计算纤维软骨分数，如果 ROI 颜色与 PF 颜色相同，则假设最大值为 1，如果 ROI 和 PF 颜色在色调饱和度颜色空间内达到最大分离度，则最小值为 -1。 <img alt="Equation 2" src="/files/ftp_upload/65801/65801eq02.jpg" style="margin: auto;"></li>
	<li>每个肢体组织切片中每个 ROI 内的色调、饱和度和纤维软骨评分的平均数据。对侧肢体组之间进行配对统计比较。</li>
</ol>9. SHG成像研究胶原网络组织的变化
<ol><li>使用带有 20 倍物镜的功能强大的显微镜系统对甲苯胺蓝染色切片进行 SHG 成像。通过截面厚度获取 z 堆栈，并根据需要执行瓷砖扫描以完全捕获跟腱插入。</li>
	<li>将SHG图像导入首选图像分析软件，并定义ROI，包括和细分跟腱插入处的CTF，如第8节中甲苯胺蓝图像分析所述（图4A，B）。</li>
	<li>如果将SHG图像导入MATLAB，请使用在MATLAB中定义CTF ROI的方法，如第8节所述。定义 ROI 后，通过将 MATLAB 中的 ROI 顶点导出为 .txt 文件并使用 文件>导入 XY 坐标导入 FIJI，将 ROI 坐标传输到 FIJI>。覆盖选择并发送给 ROI 管理器进行分析。</li>
	<li>要量化胶原蛋白的组织，请使用 FIJI 中的 Directionality 插件，该插件执行傅里叶谱分析以计算跨越 ROI 的小窗口中纤维取向的分布。这种分布的扩散称为色散，与光纤排列成反比。 注意：由于肌腱的粗大弯曲而不是缺乏对齐/组织，胶原纤维可能会在 CTF 的不同窗口中呈现出可变的方向。为了更好地区分胶原组织的变化与插入时肌腱的总弯曲，有必要定义较小的 ROI。</li>
	<li>将 SHG 图像导入 FIJI 并设置像素/长度比例。将最大像素强度数据投影到 2D 合成图像中，并将 ROI 添加到 ROI 管理器中。按顺序将每个 CTF ROI 叠加到图像上，并在 ROI 中绘制 10 个大小一致的小子 ROI，将子 ROI 添加到 ROI 管理器中。</li>
	<li>在每个子 ROI 中，运行 FIJI 中的方向性插件以计算光纤色散。每个 CTF ROI 内子 ROI 的平均离散数据，以及每个外植体各部分的每个 CTF ROI 中的平均离散数据。对侧肢体组之间进行配对统计比较。</li>
</ol>

了解肌腱撞击 – 用于研究机械生物学的小鼠外植体模型

作者没有什么可透露的。

实验性小鼠后肢外植体平台与本研究中描述的组织培养方案相结合，为研究跟腱插入处撞击驱动的纤维软骨形成的机制生物学提供了合适的模型。代表性结果证明了该外植体模型的实用性，该结果表明，在静态撞击 7 天后，甲苯胺蓝染色的显着和空间异质性变化同时维持了细胞活力。这些发现表明继发于机械撞击的富含GAG的基质分子的代谢改变，与其他模型和临床研究一致</...

许多肌腱，包括跟腱和肩袖肌腱，由于正常的解剖定位而经历骨撞击1,2,3,4.肌腱撞击产生横向指向纵向纤维轴的压缩应变5,6,7.肌腱撞击区域表现出独特的纤维软骨表型，其中收缩的圆形细胞（纤维软骨细胞）嵌入无组织的胶原网络中，糖胺聚糖 （GAG） 含量显着增加2,3,4,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24.先前的研究表明，肌腱撞击产生的不同机械环境通过驱动大型聚集蛋白聚糖（尤其是聚集聚糖）的沉积来维持这种富含GAG的基质，尽管其潜在机制尚不清楚1,3,12,13,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39.虽然纤维软骨是健康肌腱撞击区域的正常特征，但与纤维软骨形成过度相关的异常蛋白多糖代谢是肌腱病的标志特征，肌腱病是一种常见且使人衰弱的疾病，不成比例地出现在慢性撞击肌腱中1,40,41,42,43,44,45,46,47,48,49.因此，肌腱撞击在临床上被认为是驱动几种最常见的肌腱病的重要外在因素，包括肩袖疾病和插入性跟腱病 （IAT）50,51,52.目前，肌腱病的治疗效率低下。例如，大约 47% 的 IAT 患者在保守治疗失败后需要手术干预，术后结局各不相同53,54,55,56.尽管撞击和肌腱病之间存在明显的关系，但撞击肌腱细胞感知和响应其机械环境的机械生物学机制描述甚少，这模糊了对肌腱病发病机制的理解并导致治疗不充分。
外植体模型是肌腱机械生物学研究的有用工具57,58。作为了解肌腱撞击机制生物学的第一步，一些先前的研究已经探索了对细胞或切除的肌腱外植体应用简单单轴压缩后的细胞反应 27,29,30,31,32,33,34,39。然而，体外细胞缺乏细胞外和细胞周基质，这些基质可促进菌株转移，隔离机械变形释放的重要生长因子和细胞因子，并为在机械转导中发挥作用的粘着斑复合物提供底物57,59。此外，体外和切除的外植体研究都无法概括体内肌腱撞击产生的多轴机械应变环境，这取决于撞击区域的解剖学特征 5,6。在跟腱插入受撞击的情况下，这包括周围组织，例如跟骨后滑囊和 Kager 的脂肪垫 60,61,62,63。相反，肌腱撞击的体内模型 25,28,36,37,38,64,65,66 允许对直接施加到肌腱的载荷的大小和频率进行最小的控制，这是研究肌腱机械生物学的体内模型的一个公认的局限性 57,58，67,68,69,70.鉴于在体内测量肌腱应变的挑战，这些模型中产生的内部应变环境通常表征不佳。
在这篇手稿中，我们提出了一个定制的实验平台，该平台在整个小鼠后肢外植体中重建了跟骨上的跟腱插入的冲击，当与这种组织培养方案配对时，在外植体培养中保持活力超过 7 天，并允许研究肌腱撞击的生物学后遗症。该平台建立在 3D 打印的聚乳酸 （PLA） 基座上，为夹具和 3D 打印 PLA 减容插件的连接奠定了基础。握把用于将大腿和膝盖夹紧在跟腱肌腱交界处的近端，后肢的尾侧朝上，允许使用超声探头或倒置显微镜从上方对跟腱进行成像（图1A）。减容插入物沿着底座上的轨道滑动，并减少所需的组织培养基体积。缠绕在后爪上的编织线利用基础设计和 3D 打印的 PLA 夹从平台中布线。通过拉动绳子，后爪背屈，跟腱插入物撞击跟骨，导致横向压缩应变升高 5,6（图 1A）。该平台包含在丙烯酸浴中，该浴将后肢外植体浸没在组织培养基中。用胶带将绷紧的绳子固定在浴缸的外面，可以保持脚踝背屈，从而对跟腱插入产生静电冲击。3D 打印组件的 CAD 文件以多种格式提供（补充文件 1），允许导入到一系列商业和免费的开源 CAD 软件中进行修改以满足实验需求。如果无法使用 3D 打印机进行制造，则可以将 CAD 文件提供给在线 3D 打印服务，这些服务将以低成本打印和运输零件。
重要的是，肱三头肌-跟腱肌腱复合体横跨膝关节和踝关节 71,72,73。因此，跟腱的拉伸拉伤受膝关节屈曲的影响。膝关节伸展使跟腱处于紧张状态，而膝关节屈曲可降低张力。通过首先伸展膝盖，然后被动地背屈踝关节，撞击插入处的压缩应变可以叠加在拉伸应变上。相反，通过在膝关节屈曲的情况下被动地背屈踝关节，拉伸应变会减少，并且压缩应变仍然存在。目前的协议探讨了三种这样的条件。1） 对于静态撞击，足背弯曲相对于胫骨 < 110° 以撞击插入物，膝盖弯曲以减少张力。2） 对于基线张力组，踝关节伸展至背屈 145° 以上，膝关节伸展，在插入处产生主要拉伸应变。3）对于卸载组，在没有外部施加负荷的情况下，将外植体培养在培养皿中，膝盖和脚踝处于中立位置。上述角度是相对于坐标系进行照相测量的，其中足部和胫骨以 180° 的角度平行，以 90° 的角度垂直。
该方案的关键步骤包括 1） 解剖后肢外植体并小心切除皮肤和足底肌腱;2）地塞米松预处理48小时后的外植体培养;3）组织切片和组织学染色;4）彩色图像分析以评估纤维软骨的形成。解剖后，将每个后肢外植体在补充有地塞米松74的培养基中预处理48小时。将每只小鼠的对侧肢体分配到单独的实验组进行成对比较，这有助于控制生物学变异性。预处理后，将外植体如上所述放置在平台中并再培养7天（图1B）。与预处理组（第 0 天）进行额外的比较，其中在预处理 48 小时后立即去除外植体。
外植体培养后，修剪后肢，将福尔马林固定，脱钙并包埋在石蜡中。矢状面方向的连续切片提供了从肌腱交界处到跟骨插入处的跟腱的可视化，同时允许通过整个肌腱跟踪切片深度。末端脱氧核苷酸转移酶 （TdT） 介导的 dUTP X-nick 标记 （TUNEL） 用于可视化继发于细胞凋亡的 DNA 损伤并评估活力。进行甲苯胺蓝组织学和定制彩色图像分析以量化 GAG 染色的变化。然后将甲苯胺蓝染色的组织切片用于SHG成像，以表征胶原纤维组织的改变（图1B）。
提供的代表性结果表明，富含GAG的基质的组织学染色改变，模型内7天的静态撞击产生的细胞外胶原网络紊乱。该模型可用于探索撞击驱动的纤维软骨变化的分子机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbent underpads</td>
			<td>VWR</td>
			<td>82020-845</td>
			<td>For benchtop dissection</td>
		</tr>
		<tr>
			<td>Acrylic bath</td>
			<td>Source One</td>
			<td>X001G46CB1</td>
			<td>Contains the explant platform submerged in culture media</td>
		</tr>
		<tr>
			<td>Autoclave bin</td>
			<td>Thermo Scientific</td>
			<td>13-361-20</td>
			<td>Used as secondary containment, holds two platforms</td>
		</tr>
		<tr>
			<td>Base</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Braided line</td>
			<td>KastKing</td>
			<td>30lb test</td>
			<td>Used to wrap around paw and apply ankle dorsiflexion</td>
		</tr>
		<tr>
			<td>Clip</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Fisherbrand</td>
			<td>12-541-034</td>
			<td>Rectangular, No. 2, 50 mm x 24 mm</td>
		</tr>
		<tr>
			<td>Cytoseal XYL</td>
			<td>VWR</td>
			<td>8312-4</td>
			<td>Xylene-based mounting media for coverslipping Toluidine blue stained tissue sections</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>MP Biomedical LLC</td>
			<td>194561</td>
			<td>CAS#50-02-2</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), anhydrous</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>D12345</td>
			<td>CAS#67-68-5, use to solubilize dexamethasone into concentrated stock solutions</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch Brand</td>
			<td>34-8724-5195-9</td>
			<td>To attach sandpaper to Grip platens</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (1X DMEM)</td>
			<td>Gibco by ThermoFisher</td>
			<td>11965092</td>
			<td>high glucose, (-) pyruvate, (+) glutamine</td>
		</tr>
		<tr>
			<td>EDTA tetrasodium salt dihydrate</td>
			<td>Thermo Scientific Chemicals</td>
			<td>J15700.A1</td>
			<td>CAS#10378-23-1, used to make 14% EDTA solution for sample decalcifcation</td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Thermo Scientific</td>
			<td>T038181000</td>
			<td>CAS#64-17-5, 1 L supply</td>
		</tr>
		<tr>
			<td>Foam biopsy pads</td>
			<td>Leica</td>
			<td>3801000</td>
			<td>Used with processing cassettes, help hold ankle joints in desired position during fixation and decalcification</td>
		</tr>
		<tr>
			<td>Forceps, #SS Standard Inox</td>
			<td>Dumont</td>
			<td>11203-23</td>
			<td>Straight, smooth, fine tips</td>
		</tr>
		<tr>
			<td>Forceps, Micro-Adson 4.75&#34;</td>
			<td>Fisherbrand</td>
			<td>13-820-073</td>
			<td>Straight, fine tips with serrated teeth</td>
		</tr>
		<tr>
			<td>Garnet Sandpaper, 50-D Grit</td>
			<td>Norton</td>
			<td>M600060 01518</td>
			<td>Or other coarse grit sandpaper</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Chemical</td>
			<td>A38S-500</td>
			<td>CAS#64-19-7, for adjusting pH of sodium acetate buffer used for Toluidine blue histology, as well as 14% EDTA decalcification solution</td>
		</tr>
		<tr>
			<td>Grips</td>
			<td>ADMET</td>
			<td>GV-100NT-A4</td>
			<td>Stainless steel vice grips, screws and springs described in the protocol are included</td>
		</tr>
		<tr>
			<td>Histobond Adhesive Microscope Slides</td>
			<td>VWR</td>
			<td>16005-108</td>
			<td>Sagittal sections of hind limbs explants reliably adhere to these slides through all staining protocols</td>
		</tr>
		<tr>
			<td>In situ Cell Death Detection Kit, TMR Red</td>
			<td>Roche</td>
			<td>12156792910</td>
			<td>TUNEL assay</td>
		</tr>
		<tr>
			<td>Labeling tape</td>
			<td>Fisherbrand</td>
			<td>15-959</td>
			<td>Or any other labeling tape of preference</td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-100G</td>
			<td>CAS#50-81-7, for culture media formulation</td>
		</tr>
		<tr>
			<td>Neutral buffered formalin, 10%</td>
			<td>Leica</td>
			<td>3800600</td>
			<td>For sample fixation, 5 gallon supply</td>
		</tr>
		<tr>
			<td>Nunc petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P7741-1CS</td>
			<td>100 mm diameter x 25 mm height, maintain explants submerged in 70 mL of culture media as described in protocol</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100X)</td>
			<td>Gibco by ThermoFisher</td>
			<td>15140122</td>
			<td>Add 5 mL to 500 mL 1X DMEM for 1% v/v (1X) working concentration</td>
		</tr>
		<tr>
			<td>Polylactic acid (PLA) 1.75 mm filament</td>
			<td>Hatchbox</td>
			<td>-</td>
			<td>Choose filament diameter compatible with your 3D printer extruder, in color of choice.</td>
		</tr>
		<tr>
			<td>Processing cassettes</td>
			<td>Leica</td>
			<td>3802631</td>
			<td>For fixation, decalcification and paraffin embedding</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>P36931</td>
			<td>Mounting media for coverslipping tissue sections after TUNEL</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Fisher BioReagents</td>
			<td>BP1700-50</td>
			<td>CAS#39450-01-6, used for antigen retrieval in TUNEL protocol</td>
		</tr>
		<tr>
			<td>Scissors, Fine</td>
			<td>FST</td>
			<td>14094-11</td>
			<td>Straight, sharp</td>
		</tr>
		<tr>
			<td>Slide Staining Set, 12-place</td>
			<td>Mercedes Scientific&#160;</td>
			<td>MER 1011</td>
			<td>Rack with 12 stain dishes and slide dippers for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Sodium acetate, anhydrous</td>
			<td>Thermo Scientific Chemicals</td>
			<td>A1318430</td>
			<td>CAS#127-09-3, used to make buffer for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Low Profile Microtome Blades</td>
			<td>VWR</td>
			<td>25608-964</td>
			<td>For paraffin sectioning</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Thermo Scientific Chemicals</td>
			<td>348601000</td>
			<td>CAS#92-31-9</td>
		</tr>
		<tr>
			<td>Volume Reduction Insert</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Leica</td>
			<td>3803665</td>
			<td>4 gallon supply for histological staining</td>
		</tr>
	</tbody>
</table>

murine hind limb explant model for studying the mechanobiology of achilles tendon impingement

肌腱撞击骨骼会产生多轴机械应变环境，横向压缩应变明显升高，从而引发局部纤维软骨表型，其特征是富含糖胺聚糖 （GAG） 的基质积累和胶原网络的重塑。虽然纤维软骨是健康肌腱撞击区域的正常特征，但过多的 GAG 沉积和胶原网络紊乱是肌腱病的标志性特征。因此，撞击在临床上被认为是肌腱病发生和发展的重要外在因素。然而，肌腱撞击背后的机制生物学研究仍未得到充分研究。先前阐明细胞对肌腱撞击反应的努力已经对细胞施加了单轴压缩，并在 体外切除了肌腱外植体。然而，分离的细胞缺乏对机械反应至关重要的三维细胞外环境，并且 体外 和切除的外植体研究都无法概括 体内肌腱撞击产生的多轴应变环境，这取决于撞击区域的解剖学特征。此外， 肌 腱撞击的体内模型缺乏对机械应变环境的控制。为了克服这些局限性，我们提出了一种适用于研究跟腱撞击机制生物学的新型小鼠后肢外植体模型。该模型将跟腱保持在 原位 以保持局部解剖结构，并再现了在被动应用踝关节背屈期间跟腱插入跟骨上产生的多轴应变环境，同时将细胞保留在其原生环境中。我们描述了该模型不可或缺的组织培养方案，并提供了在 7 天内建立持续外植体活力的数据。代表性结果表明，组织学 GAG 染色增强，继发于撞击的胶原纤维排列减少，表明纤维软骨形成增加。该模型可以很容易地适应研究不同的机械负荷方案，并允许操纵感兴趣的分子途径，以确定介导跟腱表型变化响应撞击的机制。

A multitude of tendons, including the Achilles tendon and rotator cuff tendons, experience bony impingement due to normal anatomical positioning1,2,3,4. Tendon impingement generates compressive strain directed transversely to the longitudinal fiber axis5,6,7. Regions of tendon impingement demonstrate a unique fibrocartilage phenotype in which shrunken, round cells (fibrochondrocytes) are embedded within a disorganized collagen network with markedly increased glycosaminoglycan (GAG) content2,3,4,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. Prior studies suggest the disparate mechanical environment produced by tendon impingement sustains this GAG-rich matrix by driving the deposition of large aggregating proteoglycans, most notably aggrecan, although the underlying mechanisms are unclear1,3,12,13,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39. While fibrocartilage is a normal feature in impinged regions of healthy tendons, aberrant proteoglycan metabolism associated with excessive fibrocartilage formation is a hallmark feature of tendinopathy, a common and debilitating disease that disproportionately emerges in chronically impinged tendons1,40,41,42,43,44,45,46,47,48,49. Accordingly, tendon impingement is clinically recognized as an important extrinsic factor driving several of the most common tendinopathies, including rotator cuff disease and insertional Achilles tendinopathy (IAT)50,51,52. Currently, treatment of tendinopathy is inefficient. For example, approximately 47% of patients with IAT require surgical intervention after failed conservative management, with variable postoperative outcomes53,54,55,56. Despite the apparent relationship between impingement and tendinopathy, the mechanobiological mechanisms by which cells in impinged tendon sense and respond to their mechanical environment are poorly described, which obscures understanding of tendinopathy pathogenesis and results in inadequate treatment.
Explant models are useful tools in the study of tendon mechanobiology57,58. As a first step towards understanding the mechanobiology of tendon impingement, several prior studies have explored cellular response following the application of simple uniaxial compression to cells or excised tendon explants27,29,30,31,32,33,34,39. However, cells in vitro lack extracellular and pericellular matrices that facilitate strain transfer, sequester important growth factors and cytokines released by mechanical deformation, and provide substrate for focal adhesion complexes that play a role in mechanotransduction57,59. Additionally, both in vitro and excised explant studies fail to recapitulate the multiaxial mechanical strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region5,6. In the context of the impinged Achilles tendon insertion, this includes surrounding tissues such as the retrocalcaneal bursa and Kager&#39;s fat pad60,61,62,63. Conversely, in vivo models of tendon impingement25,28,36,37,38,64,65,66 allow minimal control over the magnitude and frequency of load applied directly to the tendon, which is a well-recognized limitation of in vivo models for studying tendon mechanobiology57,58,67,68,69,70. Given challenges in measuring tendon strain in vivo, the internal strain environment generated within these models is often poorly characterized.
In this manuscript, we present a custom experimental platform that recreates impingement of the Achilles tendon insertion upon the calcaneus within whole murine hind limb explants that, when paired with this tissue culture protocol, maintains viability over 7 days in explant culture and allows for study of the biologic sequelae of tendon impingement. The platform is built upon a 3D printed polylactic acid (PLA) base that provides the foundation for the attachment of the grips and 3D printed PLA volume reduction insert. The grips are used to clamp the upper leg and knee proximal to the Achilles myotendinous junction with the caudal aspect of the hind limb facing upward, allowing the Achilles tendon to be imaged from above using an ultrasound probe or inverted microscope (Figure 1A). The volume reduction insert&#160;slides along a track on the base and reduces the required volume of tissue culture media. A braided line wrapped around the hind paw is routed out of the platform utilizing the base design and a 3D printed PLA clip. By pulling on the string, the hind paw is dorsiflexed, and the Achilles tendon insertion is impinged against the calcaneus, resulting in elevated transverse compressive strain5,6 (Figure 1A). The platform is contained within an acrylic bath that maintains the hind limb explants submerged in tissue culture media. Securing the taut string to the outside of the bath with adhesive tape maintains ankle dorsiflexion to produce static impingement of the Achilles tendon insertion. CAD files for 3D printed components are provided in multiple formats (Supplementary File 1), allowing import into a range of commercial and free, open-source CAD software for modification to suit experimental needs. If access to 3D printers is not available for fabrication, CAD files can be provided to online 3D printing services that will print and ship the parts at low cost.
Importantly, the triceps surae-Achilles musculotendinous complex spans both the knee and ankle joints71,72,73. Consequently, tensile strain in the Achilles tendon is influenced by knee flexion. Knee extension places the Achilles tendon under tension, whereas knee flexion reduces tension. By first extending the knee and then passively dorsiflexing the ankle, compressive strains at the impinged insertion can be superimposed upon tensile strains. Conversely, by passively dorsiflexing the ankle with the knee flexed, tensile strain is reduced, and compressive strain remains. The current protocol explores three such conditions. 1) For static impingement, the foot is dorsiflexed to &#60; 110&#176; with respect to the tibia to impinge the insertion, with the knee flexed to reduce tension. 2) For the baseline tension group, the ankle is extended above 145&#176; of dorsiflexion with the knee extended, generating predominately tensile strain at the insertion. 3) For the unloaded group, explants are cultured in a Petri dish with the knee and ankle in neutral positions in the absence of externally applied load. The angles referred to above are photographically measured relative to a coordinate system where the foot and tibia are parallel at an angle of 180&#176; and perpendicular at an angle of 90&#176;.
Key steps of the protocol include 1) dissection of hind limb explants and careful removal of the skin and plantaris tendon; 2) explant culture following a 48 h dexamethasone pretreatment; 3) tissue sectioning and histological staining; and 4) color image analysis to assess fibrocartilage formation. Following dissection, each hind limb explant is pretreated for 48 h in culture media&#160;supplemented with dexamethasone74. Contralateral limbs from each mouse are assigned to separate experimental groups for pairwise comparison, which helps control biological variability. After pretreatment, explants are positioned into platforms as described above and cultured for 7 more days (Figure 1B). Additional comparisons are made to a pretreated (day 0) group in which explants are removed immediately following the 48 h pretreatment.
After explant culture, hind limbs are trimmed down, formalin fixed, decalcified and embedded in paraffin. Serial sectioning in sagittal orientation provides visualization of the Achilles tendon from the myotendinous junction to the calcaneal insertion while allowing section depth to be tracked through the entire tendon. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP X-nick labeling (TUNEL) is used to visualize DNA damage secondary to apoptosis and assess viability. Toluidine blue histology and custom color image analysis are performed to quantify changes in GAG staining. Toluidine blue stained tissue sections are then used for SHG imaging to characterize alterations in collage fiber organization (Figure 1B).
The provided representative results suggest altered histological staining of the GAG-rich matrix and disorganization of the extracellular collagen network generated by 7 days of static impingement within the model. This model can be utilized to explore molecular mechanisms underlying impingement-driven fibrocartilaginous change.

作者聚焦：揭开肌腱撞击的力学生物学 – 多轴鼠后肢外植体模型 

小鼠后肢外植体研究跟腱撞击力学生物学的模型

We study the mechanobiology underlying tendon impingement and the process by which this unique mechanical demand drives localized fiber cartilage formation in health and disease. Here we seek to characterize matrix for modeling relative to spatially heterogeneous patterns of multiaxial mechanical strain, generated by impingement and to identify molecular mechanisms mediating this response. In vitro models for studying impingement mechanobiology have applied simple compression to isolated tendon cells or artificial uniaxial compression to partial and whole tendon explants.Established animal models of tendon impingement, manipulating external source of tendon impingement in vivo most often surgically and explore biology following resuming physical activity. In vitro models present significant limitations as isolated cells lack their three dimensional extracellular environment, crucial to mechano-response. While excised explant models circumvent this limitation, both fail to recreate multiaxial strain patterns generated by impingement in vivo.Conversely, animal models offer limited ability to measure or control internal tissue strains. Our Murine Hind Limb Explant Model for studying impingement mechanobiology maintains cells within their extracellular environment and preserves local anatomy of the impinged achilles tendon insertion in situ, allowing controlled prescription of impingement through passively applied joint motion to recreate multiaxial patterns of tissue strain that are measurable and well-characterized.

TUNEL染色组织切片的代表性图像显示，在实验组的外植体培养7天后，跟腱体内的凋亡核最小（图2A）。这些图像的定量提供了证据，证明在外植体培养 7 天后，组织培养方案在不同负载条件下的外植体培养 7 天后，在跟腱内平均保持高达 78% 的活力（图 2B）。
定性地，与未加载的对照组相比，在静电冲击 7 天后，增强的甲苯胺蓝染?...

Watch this Scientific Journal Video about Unraveling the Mechanobiology of Tendon Impingement – A Multiaxial Murine Hind Limb Explant Model at JoVE.com

我们提出了一个定制的实验平台和组织培养方案，该方案以持续的细胞活力再现了由小鼠后肢外植体中跟腱插入撞击驱动的纤维软骨变化，提供了一个适合探索肌腱撞击的机制生物学的模型。

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

我们研究了肌腱撞击的机械生物学，以及这种独特的机械需求在健康和疾病中驱动局部纤维软骨形成的过程。在这里，我们试图表征矩阵，以模拟相对于撞击产生的多轴机械应变的空间异质模式，并确定介导这种响应的分子机制。用于研究撞击机械生物学的体外模型已对分离的肌腱细胞进行简单压缩，或对部分和整个肌腱外植体进行人工单轴压缩。建立了肌腱撞击的动物模型，在体内操纵肌腱撞击的外部来源，最常通过手术，并在恢复身体活动后探索生物学。体外模型存在显着的局限性，因为分离的细胞缺乏对机械反应至关重要的三维细胞外环境。虽然切除的外植体模型规避了这一限制，但两者都无法在体内重现由撞击产生的多轴应变模式。相反，动物模型测量或控制内部组织菌株的能力有限。我们用于研究撞击机制生物学的小鼠后肢外植体模型将细胞维持在其细胞外环境中，并在原位保留撞击跟腱插入的局部解剖结构，允许通过被动应用的关节运动来控制撞击处方，以重建可测量且表征良好的组织应变的多轴模式。

murine-hind-limb-explant-model-for-studying-mechanobiology-achilles

Research

JoVE Journal

Bioengineering

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

683 次观看.  University of Rochester. 我们研究了肌腱撞击的机械生物学，以及这种独特的机械需求在健康和疾病中驱动局部纤维软骨形成的过程。在这里，我们试图表征矩阵，以模拟相对于撞击产生的多轴机械应变的空间异质模式，并确定介导这种响应的分子机制。用于研究撞击机械生物学的体外模型已对分离的肌腱细胞进行简单压缩，或对部分和整个肌腱外植体进行人工单轴压缩。建立了肌腱撞击的动?...

视频: 作者聚焦：揭开肌腱撞击的力学生物学 – 多轴鼠后肢外植体模型 

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

科研

生物工程

Explant Dissection to Generate Murine Hind Limb Explant Model to Study Tendon Impingement

To begin, transfer 70 milliliters of pre-warmed Dulbecco's Modified Eagle Medium into a Petri dish in a sterile biosafety cabinet and add dexamethasone at a concentration of 100 nano molar. Assemble the surgical tools required for the dissection, which includes smooth, straight, fine tip forceps. Straight fine tip forceps with serrated teeth and straight sharp fine scissors.To dissect hind limb explants. Locate the hip joint of a euthanized mouse positioned supine on the bench top lined with absorbent under pads. Use fine scissors to create a small incision on the skin covering the proximal and anterior aspects of the upper leg.Then use fingers to pull the incision apart to expand and pinch the exposed upper leg. Carefully pull the skin distally to de-glove the hind limb to the level of the ankle. Insert one scissor blade gently under the skin along the dorsal aspect of the foot.Then make an incision that extends towards the toes and continue pulling the skin distally until it is completely removed. Position the mouse to have a clear view of the Achilles tendon insertion onto the posterior aspect of the calcaneus near the ankle. To remove the plant terrace tendon, insert one tip of the smooth fine tip forceps between the two tendons.Pass the tip medially under the plantaris tendon. Then pull it proximally to tear through the plantaris muscle. Then using fine tip serrated forceps, grasp the detached proximal end of the plantaris tendon and pull it distally to completely remove it.At the hip joint, employ fine scissors to precisely sever through the pelvis, isolating the hind limb. Use the scissors to gently remove the remaining pelvis, revealing the femoral head. In the biosafety cabinet transfer the hind limb explant into the dish containing cell culture media and dexamethasone.Relocate the dish to the incubator and subject it to pre-treatment for 48 hours.

外植体解剖生成小鼠后肢外植体模型以研究肌腱撞击

Explant Culture and Platforms Loading to Study the Biologic Sequelae of Tendon Impingement

To begin, preheat an adequate amount of sterile culture media while the 48-hour dexamethasone pretreatment of murine hindlimb explant is about to complete. Once the pretreatment ends, move the pretreated explants to the biosafety cabinet. For the unloaded group, aspirate the pretreatment media and transfer the explants to fresh Petri dishes.Add 70 milliliters of culture media to each dish before returning them to the incubator. To prepare the explant platforms for the baseline tension and static impingement group, cut pieces of sandpaper to match the size of the grip platens. For the static impingement group, cut pieces of braided line and pret tie a loose overhand knot halfway along the length of the line.Tear pieces of aluminum foil to cover each acrylic bath and spray them with 70%ethanol. Transfer the prepared components to the biosafety cabinet. Arrange the components such as acrylic bath, base, volume reduction inserts, clip, and grips for platform assembly.Place platform components inside secondary containers of sufficient volume to contain any potential culture media leaks. Submerge the containers in a solution of at least 10%bleach for a minimum of one hour. Rinse the bleach solutions with tap water.After transferring the rinsed components to the biosafety cabinet, use M3 screws and compression springs to affix the platens to the grips. Securely attach the grips to the base employing an M5 screw and insert the M6 screws until they contact the platens. Enhance the platens'functionality by affixing sandpaper using double-sided tape.Close the grips to facilitate the adhesion of the sandpaper to the platens. To load a platform, fully open the grips and use forceps to carefully position the upper leg and knee between the platens. Close the grips gently to hold the limb in place.Use forceps to grasp the exposed femoral head or foot and adjust the knee flexion angle, while gradually closing the forceps to secure it in place. For the baseline tension group, extend the knee joint while closing the grips. In the case of the static impingement group, flex the knee joint while closing the grips.For the static impingement group, place the overhand knot of the string around the distal paw and tighten it securely. Route the string through a slot in the base underneath the explant and through the clip hole. Position the base into the acrylic bath and fasten the clip to the top edge of the bath.To dorsiflex the foot, pull the string until it reaches at least 110 degrees relative to the tibia. Use a permanent marker to mark the string as it exits the clip. Capture a photograph of the explant in this dorsiflexed position for future quantification of the dorsiflexion angle.Remove the base from the setup and attach the volume reduction insert by sliding it along a track on the base. Place the base back into the acrylic bath and reposition the clip on the top edge. Use the marked string as a guide.Pull it to restore the foot to its original dorsiflexion angle and secure the string to the exterior of the bath using tape to maintain a static dorsiflexed position. For the baseline tension group, once the explant is positioned between the grips, take a photograph to quantify the dorsiflexion angle. Then slide the volume reduction insert onto the base and place it into the acrylic bath.Add 125 milliliters of prewarmed culture media to each platform to submerge the explants. After covering the top of the bath with aluminum foil, place the entire setup into a secondary container. Relocate the secondary container to the incubator and culture for seven days.