我们想感谢的材料和结构测试核心设施和工程在梅奥诊所司的技术支持。此外，我们想感谢劳伦斯 J.殷勤，詹姆斯 · 布朗，布兰特纽曼，Jorn op den Buijs，博士学位，他们在研究期间的帮助。这项研究提供了由格兰杰基金会从固安捷创新基金资助。

1.大型夹具附件 <ol><li>从机器中删除标准夹具。</li>
	<li>移动十字头分开以适应内部夹具。</li>
	<li>将铝块 （在 图 1A 部分号 1） 放在机器上和牢固地扣上使用两个螺栓机; 在中心孔可容纳机负载细胞。</li>
	<li>在铝块上放置主要夹具结构 （在 图 1B 第第 2 部分） 和安全地将它附加到块中使用 4 根螺栓。</li>
	<li>放置四吨杰克下不休息在铝块支持夹具 ( 图 1) 的夹具的一部分。</li>
	<li>在主要夹具上装载 6 信道负载细胞夹具 （ 图 1 中的部分号 3） 和安全使用 6 螺钉。</li>
</ol> 2。十字头夹具附件 <ol><li>将机十字头设置为使用十字头电梯控制的绝对零。</li>
	<li>第一基板 ( 图 2B 中的部件号 4) 重视十字头 7 螺钉用其面对的试验机前面的曲边。</li>
	<li>附加第二基板 （在 图 2 中的部件号 5） 使用一个旋转螺杆。螺丝在测试过程中容纳了左、 右的骨头。第二基板 (部件号 5) 可以自由旋转枢轴螺钉相对于第一基板 (部件号 4) 有关。第二底板的方向确定安装是否为右侧或左侧股骨。</li>
	<li>连接两个幻灯片轴承 （ 图 2D 中的部分 6 号） 第二基板 (部件号 5) 使用 4 个螺钉 （从第一基板的一侧，就可以访问两个螺钉） 的大会。旋转方式可以从第一滑床板的顶部螺丝第二套第二基板。 
	注意: 若要更改幻灯片的方向从左骨对骨，在第一基板上的四颗螺丝都松开，并对幻灯片进行旋转枢轴螺钉和再紧固在所需方向。</li>
	<li>通过将机十字头设置为 65 ° 的相对位置手动旋转幻灯片正交 6 信道负载细胞。</li>
</ol> 3。检测夹具、 高速摄像机和灯光设置实验 <ol><li>套仪器的底部夹具标准伺服液压试验机。此夹具将举行股骨和容纳左和右股骨在秋天对髋关节的配置 ( 图 1)。</li>
	<li>设置高速相机和照明设备 ( 图 3A-3D)。
	<ol><li>与机器两侧各一个三脚架上定位高强度灯光和确保它们 ( 图 3A)。</li>
		<li>为高速相机两侧的测试机和每个摄像机连接到数据采集单元 （ 图 3B -3 C） 设置三脚架。</li>
		<li>与照相机上并连接到采集单元，配置相机设置; 设置帧速率为 6000 帧 / 秒 (fps) 和分辨率为 1,024 x 512 像素; 可以降低分辨率来容纳相机内部内存 (图 3D)。</li>
		<li>集快门到 1 帧/秒 (1/6,000 fps)。此外设置相机选项，这样录音开始之前作动器移动 (100 ms 快速测试和慢测试 200 毫秒)。</li>
		<li>将两者之间的同步电缆连接相机; 在相机的软件设置中选择触发模式。</li>
	</ol></li></ol> 4。检查/标定加载细胞为适当的数据采集系统 (DAQ) <ol><li>数据采集单元设置 <ol><li>连接到测试数据采集机、 高速摄像机、 测压元件和布线中所示的线性电位器原理 图 4 中。</li>
		<li>检查正确的连接的股骨粗隆部负载细胞，磁头加载单元，线性电位器，6 信道负载细胞，并触发信号到 DAQ 设备通过观察数据信号痕迹在视图面板中的数据采集软件，通过手动推负载细胞。</li>
		<li>验证数据采集、 信号调理和脉冲发生器是所有动力。</li>
		<li>配置从负载细胞和线性电位器的所有信号的数据采集软件。在数据采集软件，选择 &#34; 一步安装 &#62; &#62; 配置 &#34; 选项卡，然后为每个与每个负载单元格关联的信号输入设置采集频率 （赫兹）。对 &#34; 触发 &#34; 选项卡上，选择适当的触发选项。视频设备也应触发期间试运行，以确保视频/数据采集系统同步性。</li>
	</ol></li> <li>额定负载 （例如至少 200 磅，最大限度地 1600 磅） 适用于股骨头和使用标准伺服液压机器验证合理负载的转子负荷细胞细胞测量和比较对制造商校准数据单 ( 图 5A)。</li>
	<li>同样，不适用于静荷载作用下 6 信道负载细胞通过使用一片死寂，如 图 5B 所示。检查功能和验证 6 信道负载电池的性能 ( 图 5A -5B) 通过计算实测和理论的力和力矩值之间的百分比差异。误差应小于 5%。 
	注: 所有负载细胞必须被都校准由其制造商提前。这一步只检查负载细胞运作、 进行所有的连接和信号是合理的。</li>
	<li>校准线性电位器 <ol><li>安全到十字头线性电位器夹具和线性电位器置于夹具 （ 图 5）。拧紧螺钉锁电位器身体和连接器插入数据采集单元</li>
		<li>手动移动 （25 毫米） 致动器上负荷帧，使电位器位置从最大压缩转换最大扩展和记录位移和相对应的电压 （至少三个数据点）。情节与电压位移和拟合数据的线性函数 (R 2 &#62; 0.95)。输入的线性方程 (m m/V) 边坡作为校正因子成 &#34; 缩放参数 &#34; 框中的数据采集软件。</li>
	</ol></li> <li>验证总体测试机安装测试代理玻璃纤维骨骨折以确保所有的数据采集功能和合理。这包括转子负荷细胞、 股骨头部测压元件、 线性电位器、 六信道负载细胞，触发信号 ( 图 6)。</li>
</ol> 5。准备测试的骨头 <ol><li>解冻在室温下 24 h 的骨头和去除水分，多余的脂肪和任何剩余的软组织使用 pa每毛巾。</li>
	<li>骨置于扫描夹具，丙烯酸和制备牙科水泥。计量 60 克 PMMA 粉和混合液体树脂在通风橱下 30 克直到粉溶解为止。混合物应浇注。为这一进程使用一次性纸杯。这一步是为灌封股骨大转子在铝杯 ( 图 7A)。</li>
	<li>对齐低于转子铝杯。然后在此基础上，倒到高度的杯中，一半的 PMMA 水泥，提高夹具平台以适合骨进杯子里。允许为聚合的 10-15 分钟。</li>
	<li>包骨头在生理盐水中的浸泡过的毛巾在骨水泥聚合期间避免组织干燥。</li>
	<li>移动到中铝杯附加到转子 ( 图 7B) 试验机测试夹具的骨</li>
	<li>中心铝杯上板附加到股骨粗隆部负载单元格，并调整幻灯片轴承，铝杯略有触动的负载单元格。从夹具旋转夹具的允许删除 pin</li> <li>中心和低与股骨头接触十字头。</li>
	<li>审查安装、 骨位置、 负载细胞信号和杯位置。此外审查 DAQ 设备;确保所有设备和负荷的细胞连接正确，并验证所有已通电。检查软件设置为适当的信号响应从每个负载细胞。</li>
	<li>股骨拍照放在从 2 面夹具。</li>
	<li>设置光圈使足够的光线到相机传感器和控制领域的深度。通过专注于股骨颈检查图像质量。这一过程应防止任何眩光和光泽骨区域图像中会影响捕获骨折事件。</li>
</ol> 6。测试到骨折 <ol><li>验证伺服机械负荷框架编程为 25 毫米的断裂试验伺服机械负荷框架中加载和卸载的适当位移控制。 
	注意: 这些是制造商特定的设置应输入和验证在控制面板的测试设备根据制造商规格。</li>
	<li>验证照明，以尽量减少反射在视频摄像机和数据采集系统，最后一次。</li>
	<li>单击开始图标从控制面板启动测试序列断裂试验股骨 ( 图 7)。</li>
	<li>从 2 边治疗股骨骨折拍照。</li>
	<li>手动收回作动器和从机删除股骨。</li>
</ol> 7。后骨折制备 <ol><li>骨去除夹具。</li>
	<li>磁带断的近端的骨轴、 包裹在湿的毛巾和塑料袋 ( 图 7)，并然后冻结在-20 ° C.</li>
	<li>保留进一步后骨折的 x 线及 CT 成像骨头。 
	注: 详细介绍了为这些进程已经从我们组 （审查在朱庇特） 以前解释在另一种协议 ( 图 7E)。</li>
</ol>

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作者有没有相关的披露。

我们提出了一项议定书，断裂测试尸体股骨近端在秋天的髋关节的配置，我们已经成功地测试了大约 200 个样本。议定书 》 包括几个内部设计的夹具为股骨强度在不同加载条件下的试验。夹具允许测试在不同的测试速度和骨方向的左边和右边股骨。后安装夹具和测量仪器，测试的玻璃纤维股骨骨折以确保所有的硬件和软件工具正确连接，同步，工作和正确地记录的信号和视频。只是之前实际尸体股骨骨折试验，盆栽在聚甲基丙烯酸甲酯的股骨干被固定在夹具。力学测试协议允许治疗股骨干骨折以可重复和一致的方式测试。
在测试期间，股骨干经验弯曲和扭转变形时的股骨头和股骨大转子被压缩了。为了避免侧向荷载的标本，十字头夹具设计两个交叉轴承允许运动在水平面上与最小的阻力。在测试过程中，这保证在股骨头部骨骼变形中的竖向荷载应用和空间运动的头。此外，这上部的夹具被为了容纳左、 右股骨通过简单地旋转板组件，如图 2所示。
底部夹具，连接到底部的试验机，被为了在测试过程中在预期内收角举行尸体股骨。这场比赛还包括测量转子和六信道负载单元连接到远端股骨测量三种力量，在轴的三个时刻的压缩载荷单轴负载细胞。此外，夹具可容纳关于虚拟点模拟膝关节股骨的旋转。
骨组织，类似于其他生物的组织，具有应变率相关的力学性能，并因此股骨强度和断裂性能将随测试速度12。因此，议定书 》 和测试夹具应该能够用于机械股测试在各种速度和容纳范围的数据采集设备、 采样频率、 高速照相机类型和照明条件。与当前的协议，我们已经成功地测试股骨在各种速度相差两个数量级 （5、 100 和 700 毫米/秒），以模仿各种创伤性事件的速度。
高速摄像机允许录制骨折一连串事件作进一步的分析。为了获得有用的数据，所有的测试组件被同步测试正确可视化将断裂力学的过程中。通过同步测压元件，位移数据和裂纹萌生和扩展数据可以分析在一起，帮助形成骨折综合图片。
为了避免挤压的大转子由于非均匀接触和不受欢迎的接触应力集中，转子被盆栽 PMMA 满杯。此外，杯子的底部是圆，使它能够在较低的夹具表面滚。这导致垂直反作用力同时防止侧向约束可能会影响断裂强度或类型的支持。本设计选择有必要获得准确的股骨强度和断裂模式类似于临床观察者。
在其他实验研究中，只有最近端股骨进行切割的股骨标本，很短的标本13通往一个主要部分。与此相反的是，当前的协议测试 255 毫米长近端股骨标本。夹具与扩展的样品长度的钢臂旨在包括近已移除的膝关节更现实地模仿侧身落在臀部的旋转点。这个延伸手臂采用 6 组件负载细胞，这用来测量三种力量，开发了在股骨干骨折测试过程中的三个时刻。这些考虑因素是类似于那些在以往的研究，描述和帮助我们更准确地、 理解促进骨折和股骨的刚度与强度14估计的力量。
3 负载导致中所获得的数据使我们能够分析力的平衡的冗余和主要的垂直方向的时刻我们夹具中的细胞。峰值转子同时生效，我们观察到非常类似震级测定不同负载细胞，平均相对误差约为 2%，这是非常令人满意的实验误差，为这一类的生物力学测试。
该协议有几个潜在的局限性。主要的限制可能的依从性夹具和试验机可以影响测量的位移和刚度15。这变得更为相关的需要更大的负荷断裂的正常股骨。然而，我们设计了我们治具厚的钢板和铝板保持刚度大于股骨刚度至少一个数量级。我们使用的约 200 股骨的样本，测量股骨刚度因夹具符合中注意平均误差为 5%左右。每个股骨纠正的刚度值然后计算校正因子。额外的潜在限制，可能会导致错误是必须严格遵循的测试步骤序列。例如，为第一的标本进行测试，保持股骨针位置之前接触到的头和转子的夹具表面未删除，而没有在远端 （固定端） 的旋转点断裂试验完成了。一个红色的长丝带附加到 pin (图 1E) 和第二个运算符以确认 pin 被测试前要求修改议定书 》。此外，测试速度时被异显著 5 700 毫米/s，我们的测试中却拟静力实验。为了深入了解股骨近端骨折加载造成的影响的较高速度下的动态行为，落塔试验可能是被雇用的16。
虽然在不同时代、 不同的运营商，执行测试，所有的股骨骨折使用相同的协议、 夹具及负载细胞，从而消除不确定性相关实验的重复性。用类似的方法，可以通过当前的协议和夹具重新设计来测试中立场配置或骨折其他骨类型。

股骨骨折风险评估的新方法及落在髋关节骨折预防发展需要对断裂过程中所涉及的生物力学过程的全面理解。尸体股骨近端强度测试已被证明是有效地确定股骨强度与股骨提供重要的见解，在此过程1，2结构承载能力影响因素之间的关系,3.实验测量股骨强度也可用于验证的基于定量计算层析成像的有限元分析 (QCT/FEA) 使断裂强度4，5，一个非侵入性的估计 6，7。
到目前为止，并无接受的标准程序来测试整个股骨标本骨折。要隔离临床可以衡量变量 （例如骨密度和几何） 和他们对股骨强度的影响，是实验测试必须以可控和可重复的方式进行。尸体股骨有不规则的形状和范围大小8中，可以索取使它无法使用内置固定装置的标准试验机测试不同年龄段的男性或女性尸体。在横盘落在髋关节事件，股骨大转子经历时股骨近端可能会遇到复杂的载荷，包括压缩、 张力、 弯矩和扭矩，压缩加载。测试这种加载场景添加实验设计的复杂性。因此，夹具，作为一个重要组成部分的测试协议，必须专门设计、 捏造，并安装以容纳股样本的不同形状和大小的不同的测试速度。此夹具也必须持有标本测试所需的方向来模拟放在臀部上坠落可能冲击载荷范围内。要满足各种各样的条件，需要有多个固定夹具和运动部件连接尽量发挥在系统中，获得平滑的荷载-位移响应的方式。
在测试过程中，可靠的数据采集也是关键的。实验设计必须纳入必要的负载细胞、 位移传感器、 信号放大器和改良剂对准确地测量力和力矩根本支持。此外，高速视频获得同步采集的部队与股骨的前部和后部意见是必要的有助于了解导致骨折，鉴定骨折类型，事件的顺序和精确定义股骨强度4，9。
虽然有宝贵的实验研究在文献中对整个股骨测试，已发布的协议要么缺乏细节上如何进行了测试，要么是非常不同的从一项研究的另一个，真正使他们重现10， 11。当前工作的目标是介绍机械测试可以作为努力的起点用于标准化测试的可重复性和可再现的骨组织的股骨样品的协议。为此，我们设计和制作测试夹具，用于测试约 200 尸体股骨。测试夹具包括底部夹具和十字头夹具。底部夹具 （图 1A-E） 举行股骨在测试过程中所需的方向，包括转子负荷细胞和连接到股骨干 6 信道负载细胞。它还可容纳三个独立的翻译，以便定位的骨骨折测试。旋转点添加到模仿膝关节。底部夹具的主要部分被组成的厚片的不锈钢和铝，使非常僵硬的夹具。负载细胞被附加到底部夹具在测试过程中测量股骨大转子上的压缩力。十字头夹具 (图 2A-2E) 包括两个铝基座和两个非常僵硬幻灯片球轴承 （连接在一起的铝板），在测试过程中占股骨头的运动，容纳为左、 右股骨。负载单元格包括在十字头夹具措施压缩力。附加到负载单元格铝杯用于将压缩荷载传递到股骨头。我们的方法用于男女各种尺寸与左、 右股骨、 颈干角，骨密度，和加载条件模仿侧身落在臀部。在我们的实验测试速度为 5、 100 和 700 毫米/秒，但他们可以设置为任何值的测试计算机上可用。设计工装夹具有两个主要组件，一个连接到测试机十字头和其他连接到测试框架。两个部分被即刻负载细胞足以测量力和力矩边界条件在所有支持。此外，两个高速摄像机被用来在测试过程中记录断裂事件。骨折后，一套 x 射线和计算层析成像 (CT) 扫描后实验性骨折分析得到了。结果从这些实验包括断裂强度和能源目前用于额外的诊断工具的研究，最终改善骨质疏松症患者近端断裂强度的评估。

<table>
	<tbody>
		<tr>
			<td>CT scanner</td>
			<td>Siemens</td>
			<td>Somatom Definition scanner (Siemens, Malvern, PA)</td>
			<td>CT scanning equipment</td>
		</tr>
		<tr>
			<td>Quantitative CT Phantom</td>
			<td>Midways Inc, San Francisco, CA</td>
			<td>Model 3 CT calibration Phantom</td>
			<td>Used for obtaining BMD values from Hounsfield units in the CT image</td>
		</tr>
		<tr>
			<td>Hygenic Orthodontic Resin (PMMA)</td>
			<td>Patterson Dental Supply</td>
			<td>H02252</td>
			<td>Controlled substance and can be purchased with proper approval</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>Kenmore</td>
			<td>N/A</td>
			<td>This is a -20oC storage for bones</td>
		</tr>
		<tr>
			<td>X-ray scanner</td>
			<td>General Electric</td>
			<td>46-270615P1</td>
			<td>X-ray imaging equipment.</td>
		</tr>
		<tr>
			<td>X-ray films</td>
			<td>Kodak</td>
			<td>N/A</td>
			<td>Used to display x-ray images</td>
		</tr>
		<tr>
			<td>X-ray developer</td>
			<td>Kodak X-Omatic</td>
			<td>M35A X-OMAT</td>
			<td>Used for developing X-ray images</td>
		</tr>
		<tr>
			<td>X-ray Cassette</td>
			<td>Kodak X-Omatic</td>
			<td>N/A</td>
			<td>Used for holding x-ray films</td>
		</tr>
		<tr>
			<td>Physiologic Saline (0.9% Sodium Chloride)</td>
			<td>Baxter</td>
			<td>NDC 0338-0048-04</td>
			<td>Used for keeping samples hydrated</td>
		</tr>
		<tr>
			<td>Scalpels and scrapers</td>
			<td>Bard-Parker</td>
			<td>N/A</td>
			<td>Used to clean the bone from soft tissue</td>
		</tr>
		<tr>
			<td>Fume Hood</td>
			<td>Hamilton</td>
			<td>70532</td>
			<td>Used for ventilation when preparing PMMA for potting of specimens</td>
		</tr>
		<tr>
			<td>Single axis load cell</td>
			<td>Transducer Techniques, Temecula, CA, USA</td>
			<td>LPU-3K; S/N 219627</td>
			<td>Capacity 3000 LBS</td>
		</tr>
		<tr>
			<td>Six channel load cell</td>
			<td>JR3,Woodland, CA</td>
			<td>45E15A4</td>
			<td>Mechanical load rating 1000N</td>
		</tr>
		<tr>
			<td>Linear potentiometer</td>
			<td>Novotechnik, Southborough, MA, USA</td>
			<td></td>
			<td>Used to acquire linear displacements during testing</td>
		</tr>
		<tr>
			<td>Slide ball bearing</td>
			<td>Schneeberger</td>
			<td>Type NK</td>
			<td>Part of the testing fixture</td>
		</tr>
		<tr>
			<td>Mechanical testing machine</td>
			<td>MTS, Minneapolis, MN</td>
			<td>858 Mini Bionix II</td>
			<td>Used for compression of femur</td>
		</tr>
		<tr>
			<td>Lighting unit</td>
			<td>ARRI</td>
			<td></td>
			<td>Needed for high speed video recordings</td>
		</tr>
		<tr>
			<td>high-speed video camera</td>
			<td>Photron Inc., San Diego, CA, USA</td>
			<td>Photron Fastcam APX-RS</td>
			<td>Used to capture the high speed video recordings of the fracture events</td>
		</tr>
		<tr>
			<td>Photron FASTCAM Viewer</td>
			<td>Photron Inc., San Diego, CA, USA</td>
			<td>Ver.3392(x64)</td>
			<td>Used to view the high speed video recordings</td>
		</tr>
		<tr>
			<td>Camera lens</td>
			<td>Zeiss</td>
			<td>Zeiss Planar L4/50 ZF Lens</td>
			<td>Needed to high image resolution</td>
		</tr>
		<tr>
			<td>Signal conditioner board (DAQ)</td>
			<td>National Instruments</td>
			<td></td>
			<td>Input/output signal connector</td>
		</tr>
		<tr>
			<td>Signal Express</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td>Data acquisition software</td>
		</tr>
		<tr>
			<td>Laptop Computer</td>
			<td>Dell</td>
			<td>N/A</td>
			<td>Used to monitor and acquire all signals from the testing procedure</td>
		</tr>
	</tbody>
</table>

method and instrumented fixture for femoral fracture testing in a sideways fall-on-the-hip position

机械测试的股骨带来有价值的见解，了解临床可以衡量的变量，例如骨矿物密度分布和几何形状对股骨力学性能的贡献。目前，尚没有用于这种措施强度和刚度的几何复杂骨头的机械试验标准协议。为了缩小这种差距我们制订了一项议定书，尸体股骨骨折并衡量其生物力学参数测试。此协议说明一套适应性强的夹具，以容纳不同的负载大小和方向为可能的骨方向在秋天会计髋关节配置，测试速度、 骨大小和左的腿右腿变化。股骨准备测试由清洗、 切割、 扫描和灌封的远端和股骨大转子接触表面在 poly(methyl methacrylate))，提出在不同的协议。准备好的标本被放置在测试夹具中模仿侧身落在臀部的位置和加载断裂。在测试期间，两个负载细胞测量垂直力适用于股骨头和股骨大转子、 六轴负载细胞测量的部队，并时刻在股骨远端和位移传感器测量差动位移之间股骨头和股骨转子区联系支持。高速摄像机被用来同步记录在测试过程中的断裂事件发生的顺序。这种数据减少允许我们进行归类的强度、 刚度，和断裂能的近 200 骨质疏松，骨质疏松，和正常的尸体股骨，为进一步发展基于工程的骨质疏松症的诊断工具的研究。

内部的夹具安装后标夹具从试验机中删除。第一，底部重夹具是安装和固定 (图 1)。这包括扩展的手臂想站 6 通道负载细胞，这也使股骨在预期内收角对齐。下一步，包括两个无摩擦滑动轴承十字头夹具安装以适应应用程序的负载和股骨头部的动作期间骨折 (图 2)。上部的夹具是可调的测试左和右腿。一旦所有夹具都安装，安装高速摄像机和照明灯具。相机中的图像测试为重点、 对比度和景深 (图 3)。所有文书然后都连接到一个数据采集单元 (图 4) 和单轴、 六轴负载单元格和线性电位器检查功能分别进行标定 (图 5)。然后测试所有文书，以确保正确的信号，从不同的负载细胞 (图 6)。股骨大转子下一步放入压克力夹具的灌封。股骨是加载到测试夹具，然后断开。骨折后股骨从夹具中移除。破碎的零件都一起录音和整个样品包裹在塑料袋里。样品是用 x 射线成像，然后用 CT 扫描为进一步骨折类型分类 (图 7)。测量的结果包括 3 部队和 3 个时刻在股骨干测量以 6 信道负载细胞和反应力在股骨头。然而，主要用于进一步 QCT/有限元分析验证结果记录在股骨大转子的力和位移记录在股骨头 (图 8)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54928/54928fig1.jpg"> 
图 1: 底夹具安装。(A) 配售安装上机器表，(B) 放置主要底部夹具结构 （第 2 部分），并确保到铝块中，(C) 主要结构的铝块 (部件号 1) 和在安全的地方，(D)整个底部夹具安装后安装 6 信道负载细胞夹具 (部件号 3) 主要的底部结构 （部分号 2）、 (E) 调整股骨干角。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54928/54928fig2.jpg"> 
图 2: 十字头夹具安装。(A) 删除夹具从机执行器，(B) 第一基板 (部件号 4) 是第一，安装 (C) 将第二基板 （部分号 5），(D) 附加两个幻灯片轴承 (部件号 6) 程序集附加到第二基板，上部的夹具; (E) 已完成安装(F) 整个测试夹具安装在机器上。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54928/54928fig3.jpg"> 
图 3: 照明和摄像机安装。(A) 设置灯和盾牌;(B) 附加高速摄像机架在三脚架上;(C) 将镜头安装到相机单元;(D) 将照相机连接到计算机。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/54928/54928fig4.jpg"> 
图 4: 示意图。与所有的输入/输出设备数据采集单元连接到数据采集。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig4large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/54928/54928fig5.jpg"> 
图 5: 校准和验证过程。检查股骨头和大粗隆力测量，(A) 单一轴负载单元格和股骨干力和力矩测量; (B) 六声道负载细胞的功能(C) 标定的线性电位器的股骨头部位移测量。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig5large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/54928/54928fig6.jpg"> 
图 6: 机械测试设置。所有文书已连接并同步机和视频照相机与沟通。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig6large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/54928/54928fig7.jpg"> 
图 7: 股骨骨折试验前后。(A) 灌封胶在铝杯股骨大转子填充与聚甲基丙烯酸甲酯;(B) 骨放置在测试夹具与股骨大转子低负载单元格上休息，而十字头夹具是联系股骨头;(C) 性断开骨右后力学测试;(D) 删除骨折股骨从机和杠子破碎的零件在一起;换行股骨在塑料袋里;(E) x 线和 CT 扫描后骨折。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig7large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/54928/54928fig8.jpg"> 
图 8: 力-位移曲线。测试在 5 和 100 毫米 s 骨折股骨的力-位移曲线。该部队被记录在大粗隆及 th在股骨头处记录电子位移。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54928/54928fig8large.jpg" target="_blank">请点击这里查看此图的大版本。</a>

Watch this Scientific Journal Video about Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position at JoVE.com

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

在这份手稿，我们提出一项议定书，在髋关节的配置使用标准伺服液压架上安装检测的夹具侧身摔倒骨折试验尸近端股骨。在测试过程中采集包括部队、 时刻和位移以及两个高速视频流的九个数字化的信号。

方法和股骨骨折测试在横盘整理的秋天在臀部位置检测的夹具

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

method-instrumented-fixture-for-femoral-fracture-testing-sideways

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Mechanical Engineering

Bioengineering

Unit 1

Force System Resultants

SCIENTISTS IN ACTION

9.9K 次观看.  Mayo Clinic. 在这份手稿，我们提出一项议定书，在髋关节的配置使用标准伺服液压架上安装检测的夹具侧身摔倒骨折试验尸近端股骨。在测试过程中采集包括部队、 时刻和位移以及两个高速视频流的九个数字化的信号。

视频: Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

一种改进的机械性能测试方法来评估骨种植体支抗

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral surface obtained from mechanical testing. This optical method requires a texture of many contrasting fiduciary marks on a solid white background for accurate tracking of surface deformation as loading is applied to the specimen. Immediately prior to testing, the surface of interest in the camera view is painted with a water-based white primer and allowed to dry for several minutes. Then, a black paint is speckled carefully over the white background with special consideration for the even size and shape of the droplets. Illumination is carefully designed and set such that there is optimal contrast of these marks while minimizing reflections through the use of filters. Images were obtained through high speed video capture at up to 12,000 frames/s. The key images prior to and including the fracture event are extracted and deformations are estimated between successive frames in carefully sized interrogation windows over a specified region of interest. These deformations are then used to compute surface strain temporally during the fracture test. The strain data is very useful for identifying fracture initiation within the femur, and for eventual validation of proximal femur fracture strength models derived from Quantitative Computed Tomography-based Finite Element Analysis (QCT/FEA).

In this protocol, the femur surface strains are estimated during fracture testing using the digital image correlation technique. The novelty of the method involves application of a high-contrast stochastic speckle pattern on the femur surface, carefully specified illumination, high speed video capture, and digital image correlation analysis for strain calculations.

基于数字图像相关的骨折检测中股骨皮质应变的估算方法

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

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

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

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

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

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

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...