这项工作得到了土木和环境工程部和伊利诺伊大学香槟分校工程学院的支持。我们承认答 Kinra 和 Kavita Kinra 奖学金, 以支持 Kavya Mendu 的研究生研究。扫描电子显微镜调查在弗雷德里克塞茨材料研究实验室和贝克曼研究所在伊利诺伊大学香槟分校的设施进行。

注: 这里描述的协议, 遵循动物保育指南的伊利诺伊州机构动物护理和使用委员会。
1. 标本采购
<ol><li>收集新鲜收获的牛股骨从美国农业部 (USDA) 认证屠宰场和运输他们在塑料空气紧密袋在一个冷却器。 注意: 在这里进行的研究中, 股骨是从 24-30 月大的动物那里采集的, 玉米喂养的, 体重约 1000-1100 磅。</li>
	<li>将股骨冻结在-20 ° c, 直到试样准备过程开始。此温度保持股骨新的12、13、14。</li>
</ol>2. 切割、清洗和嵌入试样
<ol><li>在室温下, 将冷冻的股骨在一个容器中, 用水解冻约2小时。</li>
	<li>从中部骨干区域切割多张约 10-15 毫米厚的圆盘, 使用表顶金刚石带锯生产具有均匀横截面积皮质骨的标本。</li>
	<li>使用解剖试剂盒去除皮质骨上的任何软组织或肉。</li>
	<li>切割在步骤2.2 中获得的股骨的横截面, 使用菱形切片刀片, 在湿条件下沿骨骼的纵轴进行低速锯, 以获得多个大致立方剖面。 注: 在这里, 仅对短-纵试样进行试样制备和划痕试验。但是, 除切削方向外, 其制备方法在横向方向上仍保持不变。</li>
	<li>用1.5% 阴离子清洗剂和5% 漂白水在超声波清洗机中用20分钟的时间清洗样品。</li>
	<li>
将皮质骨标本嵌入丙烯酸树脂 (聚甲基丙烯酸酯 (PMMA)) 中, 便于操作和稳定。
	<ol>
<li>要嵌入试样, 首先要在模具的壁上涂上脱模剂。然后在烧杯中混合丙烯酸树脂和固化剂, 按照 PMMA 制造商的指示。</li>
		<li>将其中一个切口皮质骨标本放入每个模子中, 表面被划伤朝下。将丙烯酸树脂混合物倒入这些准备好的标本夹中。让标本固化的时间长达 4-5 小时。</li>
	</ol>
</li>
	<li>将嵌入的标本切成5毫米厚的圆盘, 露出表面被划伤, 使用低速锯, 然后将试样安装到直径 34 mm 和高 5 mm 的金属 (铝) 圆盘上, 使用氰粘合剂。</li>
	<li>将标本包裹在汉克斯平衡盐水溶液 (HBSS) 中, 在4° c 下冷藏, 直到进一步使用1516。</li>
</ol>3. 研磨和抛光协议
注: 在小长度刻度上进行高精度测试的先决条件是平滑和平整的试样表面。以前的抛光协议13,17导致表面粗糙度很大, 导致测量的误差较大。挑战在于在大面积 3 x 8 mm2表面上实现低平均表面粗糙度, 小于 100 nm。
<ol><li>用400粒砂砾和600粒碳化硅纸分别在室温下研磨牛皮质骨标本1分钟和5分钟。保持磨床抛光机的基本速度为 100 rpm 和 150 rpm, 分别。</li>
	<li>机器研磨的牛皮质骨标本在室温下的800和1200的砂砾纸, 为期15分钟的每一步。保持磨床抛光机的基本速度为 150 rpm, 头速度为 60 rpm, 操作负载为1磅。</li>
	<li>用3µm、1µm 和0.25 µm 钻石悬浮液在硬、穿孔、无纺布上按相同顺序抛光, 室温下为90分钟。分别以 300 rpm 和 60 rpm 的抛光机的基座和头速度保持每步1磅的操作负载。</li>
	<li>使用0.05 µm 氧化铝悬浮液, 在柔软的人造人造丝布上抛光试样, 在1磅的时间内以 100 rpm 和 60 rpm 分别为90分钟, 也在室温下进行。</li>
	<li>将样品放在带有脱电离水的烧杯中, 将烧杯放入超声波浴中, 在研磨和抛光的每一步之间进行2分钟的清洗, 以清除残留物, 避免交叉污染。</li>
	<li>使用光学显微镜和 SEM 成像查看表面特征。 注意: 如图 1所示, 牛皮质骨标本上观察到了骨、哈佛运河、水泥线、间质区和空隙。这些成像方法揭示了皮质骨标本的多孔、异质性和各向异性。此外, 对试样进行了先进的表面检查, 以评估抛光表面的质量。有代表性的抛光表面显示在图 2中。</li>
</ol>4. 微划痕测试
注: 使用微划痕测试仪 (图 3) 对抛光的牛皮质骨标本进行微划痕试验。这项研究使用了一条尖端半径为200µm 的菱形罗克韦尔压和120的顶点角。该仪器允许应用线性渐进负载高达 30 N。此外, 该仪器还配备了高精度的传感器来测量水平载荷、穿透深度和由于划痕产生的声发射。该仪器可以捕捉划痕凹槽的全景图。
<ol><li>在对皮质骨标本进行测试之前, 使用聚碳酸酯作为参考材料对洛克韦尔压尖端进行标定3。</li>
	<li>将皮质骨标本放置在舞台上, 并使用光学显微镜设置到微划痕测试器模块, 选择划痕试验部位。</li>
	<li>采用线性渐进载荷, 起始载荷为 30 mN, 结束载荷为 30 N。加载速率应设置为 60 N/分钟, 划痕长度为3毫米。</li>
	<li>在短的纵向 (图 3b) 上执行一系列的划痕测试, 如图 3所示的牛皮质骨标本。</li>
	<li>湿试样表面与 HBSS 后, 每组三至四的划痕测试, 以保持他们的水合。</li>
	<li>基于非线性断裂力学模型的划痕试验数据分析2。</li>
</ol>

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作者没有什么可透露的。

微划痕测试导致混合模式的断裂3。此外, 在短的纵向牛皮质骨标本, 骨折的过程是激活的探针挖更深。对于一个3毫米长的划痕, 探测到的棱柱体积大约是3600µm 长, 600 µm 宽, 480 µm 深。这一大体积有助于预测均匀反应。一个非线性断裂力学模型, 使我们能够提取的断裂阻力基于J-积分计算1,2,4。
与早期出版物5中使用的猪标本相比, 牛皮质骨标本提供了更大的测试区域。然而, 从猪到牛皮质骨标本的显微组织特征的大小也有相应的差异。这导致开发了一个新的抛光协议的牛标本。此外, 在该方法的发展过程中, 观察到制备的牛皮质骨标本需要在准备后一周内进行测试。这是为了避免由于盐水溶液对牛标本的残留形成, 这可能会对试验结果产生严重影响。
此外, 对短的纵向牛皮质骨标本进行的试验控制了环境条件和标准化的标本制备规程。这导致了实验结果的可变性的减少从早先报告了23% 为短的纵向猪皮质骨标本5到15% 为短的纵向牛皮质骨标本在本研究。但是, 在图 7中, 逸出值测试结果可以归结为各种原因, 如盐水的贮存时间或擦伤本身的位置。然而, 鉴于骨在细观和显微长度尺度上是异质的, 预期会有一定程度的变异性。
扫描电镜显示了在这些划痕试验中骨折过程的发生率。观察了微裂纹在细观尺度上的增韧机制, 裂纹的偏转, 以及亚微米尺度下微小和纤维桥接的裂纹 (见图 5)。这与文献19中较早时报告的增韧机制是一致的。因此, 微划痕试验确定了牛皮质骨标本从中尺度到微尺度的断裂特性。
我们在这里提出的方法需要少量的标本, 并能够在较小的长度尺度上对标本进行测试。例如, 在宏观尺度上, 通过使用不同尺寸的试样, 同时具有恒定的纵横比来引入韧性到脆性转变。根据尺寸效应断裂评估技术, 至少需要5不同大小的试样来估计断裂韧性值20,21。因此, 要估计102断裂韧性值, 宏观测试需要大约510样本, 其中涉及大量的时间和资源。因此, 我们提出的方法, 以更快的速度估计断裂韧性, 是更经济的。此外, 了解不同层次层次的骨折特征, 使我们能够更有效地理解骨骼的力学。此外, 测试是有效的, 重现性的, 可以很容易地在广泛的环境控制下进行。例如, 在环境室内的盐水溶液中浸泡的试样可以进行模拟体外条件。此外, 该方法还将用于检测骨的纵向横向断裂韧性, 以获取骨的各向异性。因此, 我们的方法是一种新的手段, 骨折评估生物组织。

在本研究中, 我们用一种新颖的微划痕技术 (1、2、3、4, 测量从中尺度 (骨) 到微型 (层状) 的牛骨的断裂韧性, 5。由于不同层次的结构成分和组织不同, 断裂过程包括裂纹萌生和裂纹扩展直接影响着骨的长度尺度。因此, 评估较小长度的骨骨折是至关重要的, 以产生对骨骼脆弱性的基本认识。一方面, 常规的测试, 如三点弯曲, 紧凑的张力, 和弯曲试验通常进行的牛股骨和胫骨骨折的表征在宏观尺度6,7, 8. 另一方面, 为了测量显微尺度下的断裂韧性, 提出了硬度的压痕断裂.9。利用硬度的压进行微压痕产生径向裂纹。此外, 奥利弗法尔压断裂韧性的方法是使用一个尖锐的立方体角压10。
在上述压基断裂韧性研究中, 用观测器测量了由此产生的裂纹长度, 并用半经验模型计算了断裂韧性。然而, 这些方法是不可的, 主观的, 结果是高度依赖于观察者的技能, 由于需要测量的裂纹长度使用光学显微镜或扫描电子显微镜。此外, 在纳米级进行了划痕测试, 但基础数学模型不是基于物理的, 因为它没有考虑到由于裂缝和缺陷而导致的强度下降11。因此, 存在一个知识缺口: 一种基于物理力学模型的微观层次断裂评价方法。这一知识的缺口, 通过先聚焦于猪标本5, 促使微划痕试验应用于致密骨。这项研究现已进一步扩展, 以了解牛皮质骨。
试样的两个不同方向是可能的: 纵向横向和短纵向。纵向横向对应于股骨纵轴线的断裂性质。而短的纵向对应于股骨纵轴的断裂特性5。在这项研究中, 我们对牛皮质骨进行了划痕测试, 以表征骨在短的纵向方向上的骨折阻力。

<table><tbody><tr><td>Table Top Diamond Band Saw</td><td>McMaster Carr, Elmhurst, IL</td><td>Model&amp;nbsp; C-40</td><td>Blade speed of 40 mph; Blade dimensions: 37 inch in diameter, 0.02 inch wide and 0.14 inch deep</td></tr><tr><td>Buehler Isomet 5000 Precision Cutter</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>112780</td><td>Blade speed in the range of 200-5000 rpm in 50 rpm incrments; 8 inch diamond wafering blade</td></tr><tr><td>Branson 5800 Ultrasonic Cleanser</td><td>(Through) Grainger, Peoria, Illinois</td><td>39J365</td><td>Bransonic CPXH ultrasonic bath has a tank capacity of 2.5 gal</td></tr><tr><td>Buehler Ecomet 250 Grinder - Polisher</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>497250</td><td>8 inch base plate with a speed range from 10-500 rpm</td></tr><tr><td>Anton Paar, CSM Instruments Micro scratch tester</td><td>Anton Paar Switzerland AG</td><td>163251</td><td>Compact Platform, Acoutstic Emission Sensor</td></tr><tr><td>JEOL 6060LV general purpose scanning electron microscope</td><td>JEOL USA, Inc., Peabody, MA</td><td></td><td>Environmental scanning electron microscope which enables imaging at low vacuum levels.</td></tr><tr><td>Philips XL30 ESEM FEG&amp;nbsp;</td><td>FEI Company</td><td></td><td>Wet mode working of the instrument enables imaging of non conductive samples without altering them&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Bovine Femur</td><td>L&amp;amp;M Slaughter house, Georgetown, IL</td><td></td><td>Corn fed, 24-30 month old mature bovine specimens.</td></tr><tr><td>Alconox Powdered Precision Cleaner</td><td>Alconox, Inc., 30 Glenn St., Ste. 309, White Plains, NY, 10603</td><td>1104-1</td><td>Biodegradable, Non caustic, Interfering-residue free</td></tr><tr><td>Acrylic Plastic Casting</td><td>Electron Microscopy Sciences</td><td>24210-02</td><td>Polymethyl Methacrylate</td></tr><tr><td>CarbiMet SiC Abrasive Paper 400 grit, 8 inch, PSA backed</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>36080400</td><td>Grinding - Abrasive Papers</td></tr><tr><td>CarbiMet SiC Abrasive Paper 600 grit, 8 inch, PSA backed</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>36080600</td><td>Grinding - Abrasive Papers</td></tr><tr><td>MicroCut Discs 800 grit, 8 inch, PSA backed</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>36080800</td><td>Grinding - Abrasive Papers</td></tr><tr><td>MicroCut Discs 800 grit, 8 inch, PSA backed</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>16081200</td><td>Grinding - Abrasive Papers</td></tr><tr><td>Texmet P For 8&#039;&#039; Wheel PSA</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>407638</td><td>Polishing Cloth</td></tr><tr><td>8&#039;&#039; Microcloth PSA</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>407518</td><td>Polishing Cloth</td></tr><tr><td>Meta Di Supreme Polycrystalline Diamond Suspension, 3 &amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>406631</td><td>Polishing suspension</td></tr><tr><td>Meta Di Supreme Polycrystalline Diamond Suspension, 1 &amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>406630</td><td>Polishing suspension</td></tr><tr><td>Meta Di Supreme Polycrystalline Diamond Suspension, 0.25 &amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>406629</td><td>Polishing suspension</td></tr><tr><td>MasterPrep Polishing Suspension, 0.05&amp;micro;m</td><td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td><td>40-6377-032</td><td>Polishing suspension</td></tr><tr><td>HBSS, calcium, magnesium, no phenol red</td><td>Thermo Fisher Scientific</td><td>14025126</td><td>Buffer Solution</td></tr></tbody></table>

fragility assessment of bovine cortical bone using scratch tests

骨是一种复杂的分层材料, 具有五不同层次的组织。诸如衰老和骨质疏松等疾病等因素增加了骨骼的脆弱性, 使其容易骨折。由于社会经济对骨折的巨大影响, 我们需要有新的方法来评估每个等级的骨骼的力学性能。尽管在所有尺度下都可以探测到刚度和强度--纳米、微观、细观和宏观--断裂评估迄今仅限于宏观测试。这一局限性限制了我们对骨折的认识, 制约了实验室和临床研究的范围。在本研究中, 我们利用微划痕试验和非线性断裂力学相结合的方法, 从微观到细观长度尺度来考察骨的抗裂性。对牛皮质骨标本进行短纵向定位试验。建立了细致的实验方案, 并进行了大量 (102) 试验, 以评估皮质骨标本的断裂韧性, 同时核算与骨显微结构有关的异质性。

用原子力显微镜测量抛光表面的粗糙度。作为一个经验法则, 如果表面粗糙度是一个数量级小于感兴趣的表面特征, 那么试样就有资格作为一个抛光的。在这种情况下, 测量的表面粗糙度 60 nm 超过40µm x 40 µm 区域显然属于这个标准。
图 4显示了在短的纵向牛皮质骨标本上进行的典型划痕试验的力与穿透深度图。垂直力是规定的增量载荷, 而水平力则是探针所经受的测量阻力。图 5显示了断裂的短纵向牛皮质骨表面的扫描电镜图像。该图显示了表面的碎裂和剥落, 以及内部增韧机制的出现, 如微裂纹、裂纹偏转和裂纹桥接。基于非线性断裂力学建模2, 对微划痕试验数据进行了分析。在发生断裂过程之前, 会出现塑料耗散18。随着穿透深度的增加, 断裂过程被激活。
根据显微镜观察, 我们认为一个单一的裂纹传播, 如图 3b所示。我们建立了一个非线性断裂力学模型1,2 , 以预测划痕力的比例。在组织水平上考虑皮质骨的均匀横观各向同性组织。图 6显示了短的纵向皮质骨标本的断裂韧性的力标度。通过改变穿透深度来引入韧性-脆性过渡。在脆性和断裂驱动的制度中, 划伤力与数量成正比, 其中是探测形状函数<img alt="Equation 3" src="/files/ftp_upload/56488/56488eq3.jpg"> <img alt="Equation 1" src="/files/ftp_upload/56488/56488eq1.jpg"> 1、2、 <img alt="Equation 2" src="/files/ftp_upload/56488/56488eq2.jpg"> </img>3、4, 5。因此, 断裂韧性, 1,2,3,4, <img alt="Equation 4" src="/files/ftp_upload/56488/56488eq4.jpg"> </img>5向一个常量收敛。此外, 对应于脆性断裂的Kc 值在单个测试的强制缩放图上报告, 如图 6所示。102在短的纵向牛皮质骨标本上进行了微划痕试验, 如图 7所示。离群测试对应于在盐水溶液中经过一周的准备和贮存的试样。将试样保存很长时间, 由于盐水溶液沉淀形成了不同的断裂韧性值, 从而改变了表面。获得的整体断裂韧性值为 4.05±0.63 MPa.<img alt="Equation 5" src="/files/ftp_upload/56488/56488eq5.jpg">文献报道了断裂韧性值在2.5 到5.5 兆帕斯卡的范围内<img alt="Equation 5" src="/files/ftp_upload/56488/56488eq5.jpg">6,8。结果表明, 微划痕试验报告的断裂韧性值符合文献。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56488/56488fig1.jpg"> 图 1: 显示不同层次的骨骼标本的图表和在每个级别进行的实验性调查.水平轴对应于从宏观到纳米尺度不等的长度刻度, 垂直轴对应于每个级别的实验所对应的时间刻度。(图像信用: Kavya Mendu)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56488/56488fig1large.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/56488/56488fig2.jpg"> 图 2: (a) 用作试样底座的铝圆盘的数码照片和 (B) 抛光短的纵骨标本.<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56488/56488fig2large.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/56488/56488fig3.jpg"> 图 3: 微划痕测试.牛皮质骨标本微划痕试验的数字照片 (A)。一种罗克韦尔探针, 其顶点角为 120o探测嵌在聚甲基丙烯酸酯中的皮质骨标本。(B)划痕探针的示意图犁耕骨头材料显示短的纵向标本的混合的断裂方式的到来。(学分: 安吉蕾 Akono, 甘露 Kataruka, Kavya Mendu)。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56488/56488fig3large.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/56488/56488fig4v2.jpg"> 图 4: 划痕槽.划痕凹槽全景的光学显微图像 (A)。(B)沿划痕槽长度与深度的对应图。水平力对应于附着在微划痕试验台上的传感器检测到的电阻摩擦力, 垂直力对应于皮质骨标本上的渐进线性力。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56488/56488fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56488/56488fig5.jpg"> 图 5: 扫描电子显微镜 (SEM) 图像.扫描电镜图像的划痕凹槽显示的微观机制, 如裂纹偏转, 裂纹桥接, 纤维桥接, 并在不同的放大级别的切屑(A) 40X (B) 10,000X (C) 2,400X (D) 5,000X。使用低真空扫描电子显微镜在弗雷德里克塞茨材料科学实验室和贝克曼研究所, 伊利诺伊大学香槟分校捕获。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56488/56488fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56488/56488fig6.jpg"> 图 6: 划痕力和微划痕图像.(A)沿划痕长度的划痕力的缩放显示了断裂韧性的收敛性。是水平力, 而是依赖于几何图形和穿透深度的探测形状函数. <img alt="Equation 1" src="/files/ftp_upload/56488/56488eq1.jpg"> <img alt="Equation 3" src="/files/ftp_upload/56488/56488eq3.jpg">(B)在短的纵向方向上的牛骨上的显微划痕的全景光学显微镜图像。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56488/56488fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56488/56488fig7.jpg"> 图 7: 断裂韧性.图显示了对短的纵向牛皮质骨标本进行的102微划痕试验的断裂韧性值。<a href="//cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56488/56488fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Fragility Assessment of Bovine Cortical Bone Using Scratch Tests at JoVE.com

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

牛皮质骨脆性评估的划痕试验

本研究通过显微划痕试验, 评估了亚细层的牛皮质骨断裂韧性。这是一个原始的, 客观的, 严谨的, 和可重现的方法来探测宏观尺度下的断裂韧性。潜在的应用是研究骨质脆性的变化, 由于疾病, 如骨骼。

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

fragility-assessment-of-bovine-cortical-bone-using-scratch-tests

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.2K 次观看.  University of Illinois at Urbana Champaign. 本研究通过显微划痕试验, 评估了亚细层的牛皮质骨断裂韧性。这是一个原始的, 客观的, 严谨的, 和可重现的方法来探测宏观尺度下的断裂韧性。潜在的应用是研究骨质脆性的变化, 由于疾病, 如骨骼。

视频: Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

伤口划痕试验的优化来探测在小鼠间充质干细胞迁移的可溶性香烟烟雾提取物受损后

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

科研

发育生物学

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 ...

生物工程

Cantilever Bending of Murine Femoral Necks

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20&#176; relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6&#176; &plusmn; 1.5&#176;, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

鼠股颈悬臂弯曲

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and ...

骨折易感性机械测试的原则和方案

Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone&#39;s susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

作者聚焦：提高全骨弯曲测试的准确性和可重复性 

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

设计、执行和解释涉及啮齿动物骨骼全骨弯曲试验的研究的实际考虑因素

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care ...

<meta charset="utf8"></head><body>使用超声 BDAT 设备进行皮质骨评估

Cortical Bone Assessment Using Ultrasonic Guided Waves: A Reproducibility Study in a Healthy Population

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected to double between 2020 and 2050. Therefore, it is essential to detect patients at risk of fragility fracture at the population scale. The current golden standard is dual X-ray absorptiometry (DXA), providing the areal bone mineral density (aBMD). Ultrasonic devices, usually more portable and cheaper than X-ray devices, represent interesting DXA alternatives as screening tools. However, operator dependency is usually recognized as their main drawback. In this study, the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device is presented in detail. The dedicated ultrasonic probe is placed at the one-third distal radius of the non-dominant forearm using conventional coupling gel. The guided interface provides in quasi-real time (about 2 Hz) four parameters of interest: velocities of the first arriving signal (vFAS) and the A0 mode (vA0),&#160;cortical thickness (Ct.Th) and porosity (Ct.Po), as well as four quality parameters. The operator moves the probe slowly at the measurement site, carefully observing the feedback provided by the interface until finding a stable position and starting a series of 10 acquisitions. When at least four consistent series are obtained, the measurement ends, and an automatic report is generated. The measurement usually takes about 5 min to complete. To determine the robustness of this protocol, a reproducibility study was conducted among 3 operators (one expert and two novices) and 14 healthy participants (6 women, 8 men, 21-53 years old). The intraclass correlation coefficients (ICC) were found good for vA0 (0.76), Ct.Po (0.80) or excellent for Ct.Th (0.87) and vFAS (0.91). The standard deviations were found to be less than 10% of the total ranges in clinical practice.

<meta charset="utf8"></head><body>使用超声导波进行皮质骨评估:健康人群的可重复性研究

Here, we present the measurement protocol of the bi-directional axial transmission (BDAT) ultrasonic device in detail and test it in a reproducibility study, considering 14 healthy participants and 3 operators. Reliability, measured with the intraclass correlation coefficients (ICC), was good to excellent for four parameters of interest.

使用超声导波进行皮质骨评估:健康人群的可重复性研究

Fragility fractures are still a worldwide health burden in the context of population aging. In particular, the global number of hip fractures is expected ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

牛皮质骨脆性评估的划痕试验

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加速性骨关节炎内侧半月板和软骨划痕鼠模型的不稳定