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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 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。 2. 完全完整的关节软骨对股骨远端和肱骨近端的解剖</p…
Representative Results
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…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Calcein, AM | Invitrogen by Thermo Fisher Scientific | C3100MP | 20x50mg , Eugene, OR, USA |
| Propidium Iodide | Invitrogen by Thermo Fisher Scientific | P3566 | 1 mg/mL solution in water, 10mL, Eugene, OR, USA |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | 276855 | 1L DMSO, anhydrous, ≥99.9%, St. Louis, MO, USA |
| HBSS (calcium, magnesium, no phenol red) | Gibco by Thermo Fisher Scientific | 14025-092 | 1X, 500mL, Grand Island, NY, USA |
| Feather surgical blade (#11) | VWR | 102097-822 | Hatfield, PA, USA |
| Vapor pressure osmometer, VAPRO | ELITechGroup | Model 5520 | Puteaux, France |
| pH meter | Beckman | Model Phi 32 | Brea, CA, USA |
| Eppendorf thermomixer | Eppendorf AG | Model 5350 | Hamburg, Germany |
| Motorized inverted research microscope | Olypmus | Model IX-81 | Center Valley, PA, USA |
| Wooden applicator | Puritan Medical Products Company, LLC | 807 | 6"x100, Guilford, ME, USA |
| 1.5 Glass coverslips | Warner Instruments, LLC | 64-1696 | #1.5, 0.17mm thick, 40mm diameter, Hamden, CT, USA |
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Cite This Article
Kotelsky, A., Carrier, J. S., Buckley, M. R. Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants. J. Vis. Exp. (143), e58487, doi:10.3791/58487 (2019).