Name: real-time visualization and analysis of chondrocyte injury due to mechanical loading in fully intact murine cartilage explants
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants at JoVE.com

作者要感谢理查德·沃博士和路易斯·德尔加迪略慷慨使用了他们的 ph 计和温度计。此外, 作者还要感谢安德烈·李对机械测试系统的初步开发做出的贡献。这项研究是由国家卫生研究院 p30 ar069655 资助的。

所有的动物工作都得到了罗切斯特大学动物资源委员会的批准。
1. 解决方案
<ol><li>准备含钙、镁和无苯酚红色的汉克平衡盐溶液 (1x hbss)。通过添加少量的 hcl 或 naoh, 将 ph 值调整到7.4。</li>
	<li>通过添加氯化钠或去离子水, 将渗透度调整到 303 mosm。在解剖、样品制备和机械测试过程中使用缓冲器。</li>
</ol>2. 完全完整的关节软骨对股骨远端和肱骨近端的解剖</p...

<ol>
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上述方法成功地用于在规定的机械载荷或撞击后, 对小鼠关节的活细胞和原位关节软骨细胞损伤进行可视化。特别是, 我们能够从两个不同的滑膜关节 (膝关节 (股骨远端) 和肩部 (肱骨)) 分析完全完整的关节软骨内软骨细胞的机械脆弱性。我们具有代表性的结果表明, 关节表面细胞损伤的空间范围敏感地取决于载荷大小和冲击强度 (图 3)。重要的是, 使用这种方法有助于?...

关节软骨 (ac) 是一种承重组织, 覆盖和保护滑膜关节的骨骼, 提供光滑的关节关节关节连接。组织稳态取决于软骨细胞的活力, 软骨细胞是唯一的细胞类型居住在交流。然而, 软骨暴露于创伤 (如跌倒、车辆事故或运动损伤) 或创伤后关节不稳定造成的极端力量, 可能会导致软骨细胞死亡, 导致关节不可逆转的破裂 (骨关节炎)1. 此外, 在旨在修复受损软骨局部缺陷的骨软骨移植程序中, 移植相关的机械创伤降低了软骨细胞的活力, 并对手术结果产生不利影响2。
软骨外植体模型常用于研究关节软骨细胞对机械诱导的细胞死亡的敏感性。这些模型通常使用大型动物的外植体来研究装载条件、环境条件和其他因素对细胞脆弱性的影响3,4,5,6, 7,8,9,10,11,12, 13,14,15.然而, 由于本机接头的尺寸很大, 这些模型通常要求从完整关节的关节表面取出一个插头, 从而损害本机边界条件。此外, 它们一般需要施加较大的机械负荷来诱发细胞损伤。另外, 小鼠软骨外植体模型在研究原位软骨细胞的机械脆弱性方面提供了几个比较大的动物模型更有优势。特别是, 由于其较小的尺寸, 这些模型有助于测试完全完整的关节软骨, 而不会改变本地组织的完整性。此外, 小鼠软骨的负荷发生在小的接触区域, 这样软骨细胞死亡/损伤可以诱导小负荷 (& lt;1 n)。最后 , 小鼠基因组很容易纵 , 从而能够测试特定基因如何影响原位软骨细胞对机械损伤的易感性。
本手稿介绍的方法的总体目标是在实时中量化和可视化在体外应用机械载荷下的原位细胞死亡/损伤的空间范围。这种方法需要在不影响软骨细胞生存能力的情况下仔细解剖小鼠滑膜关节, 然后使用类似于我们最近开发的测试平台的显微镜安装设备对严重染色的外植体进行机械测试量化小鼠软骨的机械性能16。在机械测试过程中, 解剖骨的大部分 (完整) 关节表面在单个荧光显微镜上可见, 从而能够在施加负载后快速分析细胞的活力。此前对小鼠软骨外植体的表面细胞活力进行了类似的分析, 但没有同时应用负荷17。我们的方法的潜在应用包括比较研究, 以调查关节软骨细胞对不同控制的环境和机械条件的脆弱性, 以及筛选治疗方法, 以降低敏感性软骨细胞的机械负荷。

<table border="0" cellpadding="0" cellspacing="0" style="width:1027px;" width="1026">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Calcein, AM&#160;</td>
			<td style="width:205px;">Invitrogen by Thermo Fisher Scientific</td>
			<td style="width:220px;">C3100MP</td>
			<td style="width:379px;">20x50mg , Eugene, OR, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Propidium Iodide</td>
			<td style="width:205px;">Invitrogen by Thermo Fisher Scientific</td>
			<td style="width:220px;">P3566</td>
			<td>1 mg/mL solution in water, 10mL, Eugene, OR, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:205px;">Sigma-Aldrich</td>
			<td style="width:220px;">276855</td>
			<td>1L DMSO, anhydrous, &#8805;99.9%, St. Louis, MO, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">HBSS (calcium, magnesium, no phenol red)&#160;</td>
			<td style="width:205px;">Gibco by Thermo Fisher Scientific</td>
			<td style="width:220px;">14025-092</td>
			<td>1X, 500mL, Grand Island, NY, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Feather surgical blade (#11)</td>
			<td style="width:205px;">VWR</td>
			<td style="width:220px;">102097-822</td>
			<td>Hatfield, PA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Vapor pressure osmometer, VAPRO</td>
			<td style="width:205px;">ELITechGroup</td>
			<td style="width:220px;">Model 5520</td>
			<td>Puteaux, France</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">pH meter&#160;</td>
			<td style="width:205px;">Beckman</td>
			<td style="width:220px;">Model Phi 32&#160;</td>
			<td>Brea, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Eppendorf thermomixer&#160;</td>
			<td style="width:205px;">Eppendorf AG&#160;</td>
			<td style="width:220px;">Model 5350</td>
			<td>Hamburg, Germany</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Motorized inverted research microscope</td>
			<td style="width:205px;">Olypmus</td>
			<td style="width:220px;">Model IX-81</td>
			<td>Center Valley, PA, USA</td>
		</tr>
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			<td height="42" style="height:42px;width:223px;">Wooden applicator</td>
			<td style="width:205px;">Puritan Medical Products Company, LLC</td>
			<td style="width:220px;">807</td>
			<td>6&#34;x100, Guilford, ME, USA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">1.5 Glass coverslips</td>
			<td style="width:205px;">Warner Instruments, LLC</td>
			<td style="width:220px;">64-1696</td>
			<td>#1.5, 0.17mm thick, 40mm diameter, Hamden, CT, USA</td>
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real-time visualization and analysis of chondrocyte injury due to mechanical loading in fully intact murine cartilage explants

关节软骨的稳态取决于常驻细胞 (软骨细胞) 的活力。不幸的是, 机械创伤可导致广泛的软骨细胞死亡, 有可能导致关节的不可逆转的破裂和骨关节炎的开始。此外, 保持软骨细胞活力是重要的骨软骨移植程序, 以获得最佳的手术结果。我们提出了一种方法来评估细胞损伤/死亡的空间程度后, 完整的小鼠滑膜关节应用受控机械负荷或冲击。该方法可用于比较研究, 以研究不同的机械加载方案, 不同的环境条件或遗传操作, 以及不同阶段的软骨退化的短期或长期的影响原位关节软骨细胞的脆弱性。该协议在手稿中介绍的目的是评估小鼠滑膜关节关节表面细胞损伤死亡的空间程度。重要的是, 这种方法能够在不影响本地边界条件的情况下对完全完整的软骨进行测试。此外, 它还允许实时显示严重染色的关节软骨细胞, 并基于单一图像分析的细胞损伤的应用受控静态和冲击加载方案。我们具有代表性的研究结果表明, 在健康软骨外植体中, 细胞损伤的空间程度敏感地取决于负荷大小和冲击强度。我们的方法可以很容易地适应研究不同的机械加载方案, 不同的环境条件或不同的遗传操作对原位关节软骨细胞的机械脆弱性的影响。

6种不同的应用加载方案 (静态加载: 0.1 n、0.5 n 和 1 n, 5分钟; 冲击载荷: 1 mj、2 mj 和 4 mj) 可重复诱导可量化的可量化的股骨和肱骨软骨细胞损伤局部区域, 这些区域是从8-10 的 bb/c 小鼠身上获得的 (图 2)。重要的是, 在 imagej 中快速、方便地测量了关节表面软骨细胞损伤的空间范围。研究结果表明, 关节软骨细胞的机械脆弱性受载荷大小和冲击强度的影响...

Watch this Scientific Journal Video about Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants at JoVE.com

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

全完整型小鼠软骨外种机械负荷对软骨细胞损伤的实时可视化与分析

我们提出了一种方法来评估细胞损伤死亡的空间程度后, 完整的小鼠关节关节应用受控机械负荷或冲击。该方法可用于研究骨关节炎、遗传因素和不同的加载方案如何影响原位软骨细胞的脆弱性。

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

这种方法能够调查不同机械载荷方案、环境条件或软骨退化阶段对原位关节软体细胞脆弱性的影响。物理力。与其他技术不同，这些测量是在完全完整的软骨中实时进行的，同时不影响原生边界条件。这项技术的另一个优点是它使用小鼠模型，允许测试特定基因如何影响原位软体细胞在机械损伤中的易感性。对于深骨股骨的解剖，将鼠标放在超活位置，并在膝盖前部进行五毫米的皮肤切口。将切口一直伸到膝盖周围。并拉回皮肤暴露膝盖关节和腿部肌肉。从股骨的近端开始，使用11号手术刀刀片沿远端方向切割骨骼。将刀片定位在四头肌肌腱单元和股骨轴前侧之间。将切口伸过帕泰拉。切穿肌腱中间切掉四头肌肌腱单元。接下来，从股骨的近端开始，将手术刀刀片定位在腿筋肌肉肌腱单元和股骨轴的后侧之间，并沿着骨骼沿远端方向切割。当切口接近膝关节时，切开软组织，完成膝盖关节的切口。拉回四头肌和腿筋肌肉，露出股骨。切掉骨的侧侧和中侧多余的肌肉。切开近端侧小腿肌肉。翻转腿以可视化股骨的后侧。取出膝关节周围的多余的组织，露出股骨和近侧头骨的后表面的侧孔。切开前十字韧带和后十字韧带，远离股骨。将头骨从股骨上拉开，切开所有韧带，将小腿与股骨分开。使用标准的解剖剪刀，从侧侧切穿骨骼近端的股骨，比侧侧骨关节高出约八毫米。并使用珠宝商的钳子从股骨上去除周围的软组织。然后将软骨暴露在股骨端端的两个圆锥体上。定期用HSSS水合关节表面。对于腐殖质的解剖，请将鼠标放在超活位置。用微型剪刀在肘部后侧做五毫米切口。伸展肘部周围的切口，拉回皮肤，露出手臂和肩部的肌肉。从腐殖质的近端开始，将十一号手术刀的刀片在三头肌肌腱单元和腐殖质的后侧之间放置。沿着骨头沿着小方向切开。通过三头肌肌腱向腐殖质的端端延伸切口，将三头肌拉回腐殖质的近端，直到腐殖质头部暴露。在不接触关节表面的情况下切割腐殖质头部周围的结缔组织。从身体里取下肢体用HSSS水合腐殖质头的关节表面。要断开腐殖质与手臂的连接，请使用钳子切断手臂后侧的 ulna 的近端。切开腐殖质端周围的结缔组织，去除骨骼上多余的组织。然后使用标准解剖剪刀去除腐殖质后侧的三叶虫管。将解剖的标本放入含有HSS的1.5毫升微离心管中。对于样品的钙素 AM 染色，将骨骼转移到 HBSS 中含有 500 微升的 10.05 微摩尔钙化物的 1.5 毫升微离心管中。在 800 rpm 时将管以 37 摄氏度的高温混合器中放入热混合器中，防止光线。三十分钟后，用新鲜的HBSS清洗标本十分钟。将试样转移到安装在定制显微镜的玻璃幻灯片上，安装机械测试装置，使后股心结的关节表面或腐殖质头部坐在玻璃上。使用 HBSS 使试样水合，将带试样的设备置于荧光显微镜阶段。在负载应用之前，在 4 倍放大下对沾有钙素 AM 的关节软骨细胞进行成像。调整采集设置以优化图像质量。要施加静态机械载荷，请将规定的静态负载放在试样顶部，以便将关节软骨压缩在盖玻璃上五分钟，然后拆下负载。要施加冲击机械负载，请从规定高度将已知重量的圆柱形冲击器滴到试样上，在撞击后五秒钟释放负载。去除任一负载后，立即在一毫升HSS中孵育新准备的60微摩尔丙酸碘化物中的标本。在室温下五分钟，然后通过荧光显微镜重新成像关节软骨细胞，正如刚刚演示的。为了量化由于应用机械载荷方案而导致的损伤细胞和死亡细胞的面积，首先打开在 ImageJ 中应用机械载荷之前和之后获得的关节表面的显微图。将图像合并到堆栈中。根据图像分辨率设置图像比例。接下来，定义细胞成为钙素阴性且PI阳性的区域。这些细胞被认为是受伤或死亡。然后使用测量工具确定受伤死细胞的面积。至少有六个测试应用了加载协议，在股骨和腐殖质的骨质损伤中可重复诱导可量化的局部区域，从8至10周大的瓣膜C小鼠获得。在测试的所有加载协议中，更高的负载幅度和更高的冲击强度显著加剧了股骨和腐殖质细胞损伤的种族程度。执行解剖时，必须记住不要与手术工具接触关节软骨，因为这可能通过标本的能力而损害基线。使用这种镜像模型可能有助于骨关节炎的研究，因为这种疾病很容易通过遗传、饮食或手术操作在小鼠中诱发。我们的平台还提供一个工具，用于询问基础科学问题，并针对软细胞的机械脆弱性筛选治疗干预措施。

Research

JoVE Journal

Bioengineering

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

科研

生物工程

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Test Bed to Examine Helmet Fit and Retention and Biomechanical Measures of Head and Neck Injury in Simulated Impact

测试床, 检查头盔的拟合和保留, 以及在模拟撞击时头颈部损伤的生物力学措施

Conventional wisdom and the language in international helmet testing and certification standards suggest that appropriate helmet fit and retention during ...

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

在微生物发酵过程中制造一种纳菲安涂层、还原石墨烯 Oxide/Polyaniline 化学电阻传感器, 以实时监测 ph 值

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

预测神经植入物软化行为的环境动力力学分析

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

阳性肽载体软骨内运输特性的特征

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

实时活体内多光子显微镜可可视化聚焦超声和微气泡处理，以提高血脑屏障通透性

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...