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Bioengenharia

基于原子力显微镜的人关节软骨外植体微压痕研究的实际问题

Published: October 28, 2022
doi:
10.3791/64371
, , ,
1Laboratory of Cell Biology, Department of Orthopaedic Surgery,University Hospital Tübingen, 2Department of Oral and Maxillofacial Surgery,University Hospital Tübingen

Summary

我们提出了一种循序渐进的方法来识别和解决与原子力显微镜微压痕相关的最常见问题。我们举例说明了以不同程度的骨关节炎驱动的变性为特征的天然人类关节软骨外植体的新问题。

Abstract

毫无疑问，原子力显微镜（AFM）是目前评估生物领域微观甚至纳米线索的最强大和最有用的技术之一。然而，与任何其他微观方法一样，可能会出现方法学挑战。特别是，样品的特性、样品制备、仪器类型和压痕探头都可能导致不必要的伪影。在该协议中，我们举例说明了健康和骨关节炎关节软骨外植体的这些新问题。为此，我们首先通过分步方法展示如何 通过 整个组织外植体的大型 2D 镶嵌荧光成像，根据变性的不同阶段生成、分级和视觉分类离 体 关节软骨盘。 离体 模型的主要优势在于它由老化的、天然的、人类软骨组成，可以研究骨关节炎从早发到进展的相关变化。此外，还介绍了组织制备中的常见陷阱，以及实际的AFM程序以及随后的数据分析。我们展示了样品制备和处理、高级退化引起的形貌样品特性以及样品-尖端相互作用等基本但关键的步骤如何影响数据采集。我们还仔细研究了AFM中最常见的问题，并在可能的情况下描述了如何克服这些问题。了解这些局限性对于正确的数据采集、解释以及最终将研究结果嵌入到广泛的科学背景中至关重要。

Introduction

由于电子设备和系统的尺寸不断缩小，微纳基技术和装备的快速发展势头强劲。其中一种设备是原子力显微镜 （AFM），它可以扫描生物表面并在纳米和微米尺度上检索地形或生物力学信息1,2。在其广泛的功能中，该工具可以作为微压头和纳米压头操作，以获取有关各种生物系统的机械性能的信息3,4,5,6。数据是通过机械探针与表面的物理接触来收集的，机械探针的尖端可以小至约 1 nm7。然后根据悬臂尖端的压痕深度和施加在样品上的力8 显示样品的变形。

骨关节炎（OA）是一种长期退行性慢性疾病，其特征是关节和周围组织中的关节软骨恶化，这可能导致骨表面完全暴露。OA的负担是沉重的;目前，65岁及以上的女性中有一半和男性中有三分之一患有OA9。创伤、肥胖和由此导致的关节10 生物力学改变决定了关节软骨退化，这被视为常见的最终结果。Ganz 等人的开创性研究认为，OA 过程的早期步骤可能涉及软骨的生物力学特性11，从那时起，研究人员证实了这一假设12。同样，人们普遍认为，组织的生物力学特性在功能上是由超微结构组织以及细胞-细胞和细胞-基质串扰协调的。任何改变都会极大地影响整体组织的生物力学功能13。迄今为止，OA 诊断是临床诊断，并且基于平片 X 线照相14。这种方法是双面的：首先，缺乏明确的退行性临界阈值来制定 OA 的诊断，使得病情难以量化，其次，成像方法缺乏敏感性和标准化，无法检测局部软骨损伤15,16,17。为此，软骨机械性能的评估具有决定性的优势，即它描述了在OA过程中变化的参数，无论疾病的病因如何，并且在非常早期的阶段对组织功能有直接影响。压痕仪器测量组织抵抗压痕的力。事实上，这并不是一个新概念;最早的研究可以追溯到 1980 年代和 1990 年代。在此期间，大量研究表明，设计用于关节软骨关节镜测量的压痕仪器可能非常适合检测软骨的退行性变化。甚至在 30 年前，一些研究就能够证明压痕仪器能够通过在关节镜检查期间进行压缩刚度测量来检测组织变性过程中软骨表面的体内变化18,19,20。

关节软骨的 AFM 压痕 （AFM-IT） 提供有关组织的关键机械特性（即刚度）的信息。这是一个机械参数，描述了施加的无损载荷与缩进组织区域21的变形之间的关系。AFM-IT 已被证明能够量化宏观上未受影响的胶原网络中僵硬的年龄依赖性变化，从而区分与 OA 发病相关的病理变化（关节软骨外桥量表 0 级）22。我们之前已经证明，AFM-ITs基于空间软骨细胞组织作为早期软骨退行性变的基于图像的生物标志物，不仅可以量化，而且可以实际确定最早的退行性机械变化。这些发现已经得到其他人的证实 23,24.因此，AFM-IT是诊断和识别早期退行性变化的有趣工具。这些变化已经可以在细胞水平上测量，重塑对OA病理生理过程的理解。

在该协议中，我们展示了关节软骨外植体的完整组织学和生物力学分级程序，从天然软骨外植体制备到 AFM 数据采集和处理。通过循序渐进的方法，我们展示了如何通过二维大马赛克成像，然后进行微AFM压痕，根据变性的不同阶段对关节软骨组织进行生成、分级和视觉分类。

尽管目前，AFM-IT 是测量软骨生物力学变化的最灵敏的工具之一7，但与任何其他仪器技术一样，它具有局限性和实际特性25，可能导致错误的数据采集。为此，我们仔细研究了软骨外植体AFM测量过程中出现的最常见问题，并在可能的情况下描述了如何最大限度地减少或克服这些问题。这些因素包括样品的形貌方面和在与AFM兼容的环境中稳定它们的困难，组织表面的物理特性，以及在这些表面上进行AFM测量的困难。还给出了错误的力-距离曲线的示例，强调了可能导致它们的条件。还讨论了悬臂尖端几何形状固有的其他限制以及使用赫兹模型进行数据分析。

Protocol

使用从德国图宾根大学医院接受全膝关节置换术的患者身上采集的股骨髁。本研究仅包括来自退行性和创伤后关节病变患者的关节软骨样本。在研究开始之前，已获得部门、机构和地方伦理委员会的批准（项目编号：674/2016BO2）。在参与之前，已收到所有患者的书面知情同意书。 注：图1中给出了按时间顺序排列的实验步骤的流程 图。 <p class="jove_title…

Representative Results

使用自制的切割装置，我们能够从新鲜的人髁突中取出并生成小（4 mm x 1 mm）软骨盘，其中包含单细胞空间模式30，包括单串（SS，图2A），双串（DS），小簇（SC），大簇（BC;图2A）和漫反射（图2B）。具有代表性的软骨外植体如图3A所示。使用自上而下的荧光成像选择仅显示一种类型图案的?…

Discussion

作为一种进行性和多因素疾病，OA会引发关节软骨的结构和功能变化。在整个 OA 过程中，机械特征的损伤伴随着关节软骨表面的结构和生化变化27,31。OA中最早发生的病理事件是蛋白多糖耗竭伴胶原网络破坏32,33,34。这种早期的细微表面变化很难通过批量测试来精确定位和识别，因为?…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢图宾根大学医院骨科的骨科医生提供组织样本。

Materials

Amphotericin B Merck KGaA, Darmstadt, Germany 1397-89-3
Atomic force microscop (AFM) head  CellHesion 200, Bruker Nano GmbH, Berlin, Germany JPK00518
Biocompatible sample glue  Bruker Nano GmbH, Berlin, Germany H000033
Calcein AM Cayman, Ann Arbor, Michigan, USA 14948 Cell membrane permeable stain, used for cartilage disc sorting- top view imaging
Cantilever Bruker Nano GmbH, Berlin, Germany SAA-SPH-5UM Frequency Nom: 30KHz, k: 0.2N/m, lenght nom: 115μm, width nom: 40μm,  geometry: rectangular, cylindrical tip with a 5μm end radius
Cartilage ctting device  Self-made  n/a Cutting plastic device containing predefined wholes of 4mmx1mm
CDD camera integrated in the AFM The Imaging Source Europe GmbH, Bremen, Germany DFK 31BF03
CDD camera integrated in the fluorescence microscope Leica Biosystems, Wetzlar, Germany DFC3000G
Cryotome Leica Biosystems, Wetzlar, Germany CM3050S 
Data Processing Software for the AFM Bruker Nano GmbH, Berlin, Germany n/a Version 5.0.86,  can be downloaded for free from the following website https://customers.jpk.com
Dulbecco's modified Eagle's medium (DMEM)  Gibco, Life Technologies, Darmstadt, Germany 41966052
Fluorescence Microscope (Leica DMi8) Leica Biosystems, Wetzlar, Germany 11889113
Glass block cantiliver holder Bruker Nano GmbH, Berlin, Germany SP-90-05 Extra long glass block with angled faces, designed especially for the use with the JPK PetriDishHeaterTM (Bruker).
Inverted phase contrast microscope (integrated in the AFM) AxioObserver D1, Carl Zeiss Microscopy, Jena, Germany L201306_03
Leibovitz's L-15 medium without L-glutamine  Merck KGaA, Darmstadt, Germany F1315
Microscope glass slides Sigma-Aldrich, St. Louis, Missouri, USA CLS294775X50
Mounting medium With DAPI ibidi GmbH, Gräfelfing, Germany 50011 Mounting media with nuclear DAPI (4′,6-diamidino-2-phenylindole) counterstaining used for cartilage discs  side view imaging
Penicillin-Streptomycin Sigma-Aldrich, St. Louis, Missouri, USA P4333
Petri dish heater associated with AFM (Petri Dish Heater) Bruker Nano GmbH, Berlin, Germany T-05-0117
Scalpel Feather Medical Products, Osaka, Japan 2023-01
Silicone Skirt Bruker Nano GmbH, Berlin, Germany n/a Protective silicone membrane (D55x0.25) which is placed on the basis of the base of the glas block to prevent  medium condensation in the AFM head.
Statistical program – SPSS IBM, Armonk, New York, USA SPSS Statistics 22 Vesion 280.0.0.0 (190)
Tissue culture dishes  TPP Techno Plastic Products AG, Trasadingen, Switzerland TPP93040
Tissue-tek O.C.T. Compound Sakura Finetek, Alphen aan den Rijn, Netherlands SA6255012 Water-soluble embedding medium 
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Tags

Keywords: Articular CartilageOsteoarthritisMicro-indentationAtomic Force MicroscopyCartilage ExplantsBiomechanical PropertiesIn Vitro ModelCartilage DiscFluorescence MicroscopyAFM Calibration
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Daniel, C., Alexander, D., Umrath, F., Danalache, M. Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants. J. Vis. Exp. (188), e64371, doi:10.3791/64371 (2022).
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