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。

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			<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&#160;</td>
			<td>FEI Company</td>
			<td></td>
			<td>Wet mode working of the instrument enables imaging of non conductive samples without altering them&#160;</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Femur</td>
			<td>L&#38;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&#39;&#39; Wheel PSA</td>
			<td>Buehler,41 Waukegan Rd, Lake Bluff, IL 60044</td>
			<td>407638</td>
			<td>Polishing Cloth</td>
		</tr>
		<tr>
			<td>8&#39;&#39; 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 &#181;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 &#181;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 &#181;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&#181;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

编制完整的牛尾巴器官培养的椎间盘

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

在心肌结构，功能的体内定量评估，灌注与活力使用心脏微型计算机断层扫描

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Modified Drop Tower Impact Tests for American Football Helmets

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards is presented that would provide better assessment of a helmet's on-field impact performance by including a faceguard on the helmet. In this study, a merger of faceguard and helmet test standards is proposed. The need for a more robust systematic approach to football helmet testing procedures is emphasized by comparing representative results of the Head Injury Criterion (HIC), Severity Index (SI), and peak acceleration values for different helmets at different helmet locations under modified NOCSAE standard drop tower tests. Essentially, these comparative drop test results revealed that the faceguard adds a stiffening kinematic constraint to the shell that lessens total energy absorption. The current NOCSAE standard test methods can be improved to represent on-field helmet hits by attaching the faceguards to helmets and by including two new helmet impact locations (Front Top and Front Top Boss). The reported football helmet test method gives a more accurate representation of a helmet's performance and its ability to mitigate on-field impacts while promoting safer football helmets.

This article provides a novel technique to assess the performance characteristics of American football helmets by inclusion of faceguards during NOCSAE Standard drop tests. Additionally, two more impact locations are proposed to be added to the NOCSAE certification.

修改落塔冲击试验为美国橄榄球头盔

A modified National Operating Committee on Standards for Athletic Equipment (NOCSAE) test method for American football helmet drop impact test standards ...

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 ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生生物材料的生物相容性曲线

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

兔子骨增增的千叶模型,用于骨代材料骨生长和新血管化的评估

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

使用双平面影像学测量 3D 体内肩部运动学

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

用于工程神经样组织的小鼠皮质星形胶质细胞的3D生物打印

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

3D 共培养检测:用于癌症药物筛选的血管化成骨生态位

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

作者聚焦: 在成像微孔板中使用预制聚乙二醇 （PEG） 水凝胶简单建立血管化成骨骨髓生态位  

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

在成像微孔板中使用预制聚乙二醇（PEG）水凝胶简单建立血管化成骨骨髓壁龛

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...