一种摩擦试验-用于研究滑膜关节生物力学、机械生物学和物理调节的生物反应器装置
Summary
本方案描述了一种摩擦测试装置，该装置将同时相互滑动和正常载荷施加到两个接触的生物配合面上。
Abstract
在原发性骨关节炎（OA）中，与衰老相关的正常“磨损”会抑制软骨维持其承重和润滑功能的能力，从而促进有害的物理环境。关节软骨和滑膜的摩擦相互作用可能通过组织水平磨损和细胞机械转导影响关节稳态。为了研究这些机械和机械生物学过程，描述了一种能够复制关节运动的装置。摩擦测试装置控制向两个接触的生物配合面传递往相互平移运动和正常载荷。本研究采用滑膜软骨配置，并在磷酸盐缓冲盐水（PBS）或滑液（SF）浴中进行测试时提供摩擦系数测量结果。该测试针对一系列接触应力进行了测试，突出了SF在高负载下的润滑性能。该摩擦测试设备可用作仿生生物反应器，用于研究活关节组织的物理调节，以响应与双关节关节关节相关的应用生理负荷。
Introduction
骨关节炎（OA）是一种使人衰弱的退行性关节疾病，影响超过3200万美国成年人，医疗保健和社会经济成本超过165亿美元1。该疾病的典型特征是关节软骨和软骨下骨的降解;然而，滑膜的变化最近引起了人们的赞赏，因为滑膜炎与OA症状和进展有关2，3，4。在原发性（特发性）OA中，与衰老相关的正常“磨损和撕裂”会抑制软骨维持其承重和润滑功能的能力。关节软骨层长时间滑动接触或软骨与植入材料滑动接触所产生的应力已被证明可以通过地下疲劳失效促进分层磨损5，6。由于关节7，8中存在动态机械环境，关节软骨和滑膜的摩擦相互作用可能通过组织水平磨损和细胞机械转移来影响关节稳态。为了研究这些机械和机械生物学过程，设计了一种装置来复制关节的运动，并严格控制压缩和摩擦载荷5，6，9，10，11，12，13。
本方案描述了一种摩擦测试设备，该设备将相互平移的运动和压缩载荷传递到活组织外植体的接触表面。计算机控制的设备允许用户控制每次测试的持续时间、施加的载荷、平移台的运动范围以及平移速度。该设备是模块化的，允许测试各种配合面，例如组织上的组织（软骨上的软骨和软骨上的滑膜）和玻璃上的组织。除了测试仪获得的功能测量外，还可以在测试之前和之后评估组织和润滑浴组件，以评估给定实验方案赋予的生物变化。
软骨摩擦学的研究已经进行了几十年，并且已经开发了几种技术来测量软骨与玻璃和软骨之间的摩擦系数，软骨14，15。不同的方法是由连接的和/或感兴趣的润滑机制驱动的。在实验变量的控制和生理参数的概括之间经常存在权衡。摆式装置利用完整的关节作为简单摆锤的支点，其中一个关节表面在第二个表面上自由平移14，16，17，18。摩擦测量不是使用完整的关节，而是通过在所需表面上滑动软骨外植体14，19，20，21，22，23，24，25来获得摩擦测量。据报道，关节软骨的摩擦系数在很宽的范围内（从0.002到0.5）变化，具体取决于操作条件14，26。已经创建了复制旋转运动的设备23，27，28。Gleghorn等人.26开发了一种多孔定制摩擦计，以使用Stribeck曲线分析观察软骨润滑曲线，并在软骨之间相对于平板玻璃配合面施加线性振荡滑动运动。
该装置旨在隔离摩擦反应，并探索各种载荷条件下活组织的机械生物学。该装置采用简化的测试设置，通过压缩滑动模拟关节关节关节，可以近似滚动和滑动运动，并了解纯滚动运动中的阻力相对于关节软骨29的测量摩擦系数可以忽略不计。该测试仪最初是为了研究间质液加压对关节软骨9摩擦反应的影响而构建的，此后一直用于探索诸如去除软骨浅表区域的摩擦效应10，滑液11的润滑作用，软骨磨损假设5，6，30和滑膜对组织摩擦测量13等主题。.摩擦测试生物反应器可以在无菌条件下进行摩擦实验，为探索摩擦力如何影响活软骨和滑膜的机械生物学反应提供了一种新的机制。该设计可用作仿生生物反应器，以研究活关节组织的物理调节，以响应与双关节关节关节相关的应用生理负荷。
本研究提出了一种在一系列接触应力和不同润滑浴中进行滑膜软骨摩擦测试的配置。大多数关节的关节表面积在很大程度上是滑膜组织31。虽然滑膜在软骨上滑动不会发生在初级承重表面，但两个组织之间的摩擦相互作用可能仍然对组织水平修复和细胞机械转导具有重要意义。先前已经表明，位于滑膜内膜层上的成纤维细胞样滑膜细胞（FLS）具有机械敏感性，对流体诱导的剪切应力32作出反应。还证明拉伸33、34 和流体引起的剪切应力35 可调节 FLS 润滑剂的产生。因此，滑膜和软骨之间的直接滑动接触可以为滑膜中的常驻细胞提供另一种机械刺激。
只有少数关于滑膜摩擦系数的报告已经发表31，36。Estell等人.13 试图通过利用生物学相关的配合面来扩展先前的表征。由于摩擦测试设备能够测试活组织，因此可以模拟关节关节连接过程中的生理组织相互作用，以阐明接触剪切应力对滑膜细胞功能的作用及其对滑膜和软骨之间串扰的贡献。后者与关节炎和损伤后滑膜关节炎症的介导有关。由于软骨与滑膜和滑液的物理接近，滑液含有表现出多能能力的滑膜细胞，包括软骨发生，因此假设滑膜细胞通过植入关节表面在软骨稳态和修复中发挥作用。在这种情况下，软骨 -滑膜和滑膜-滑膜的物理接触和相互剪切可能会增加滑膜细胞对软骨损伤区域的可及性37，38，39，40。利用滑膜软骨配置的研究不仅可以深入了解关节大组织力学和摩擦学，还可以带来维持关节健康的新策略。
Protocol
从当地屠宰场获得的幼年牛膝关节用于本研究。具有此类牛标本样本的研究不受哥伦比亚机构动物护理和使用委员会（IACUC）的约束。 1. 摩擦试验装置的设计 注：摩擦测试装置的示意图如图 1所示。该装置建在刚性底板上（未显示），底板用作结构支撑的平台。 将步进电机连接到水平平移台（参见 材料表<…
Representative Results
软骨上的滑膜结构用于摩擦测试幼年牛外植体。滑膜安装在直径为10毫米的丙烯酸加载压板上，使得内膜层将与下面的软骨接触。胫骨条用作软骨配合面（图6A）。胫骨条的切割深度约为1.4 mm，尺寸为10 mm x 30 mm。将样品在37°C下在磷酸盐缓冲盐水（PBS）浴或牛滑液（SF）浴中测试1小时。SF浴由PBS和牛SF的50/50混合物组成。载物台加速度为100 mm/s2，载物台速度为1 mm/s，载?…
Discussion
关节内存在动态机械环境，因为软骨受到压缩，拉伸和剪切力，以及流体静压和渗透压44，45。虽然软骨是关节的主要承重组织，但滑膜也与软骨表面和自身在组织折叠的区域发生摩擦相互作用。软骨和滑膜之间的物理相互作用可能负责转移细胞并将间充质干细胞释放到关节环境中，提供潜在的细胞来源，以促进（有限的）关节软骨修复机制<sup class="xref…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了骨科科学研究基金会，NIH 5R01 AR068133，NIH TERC 5P41EB027062和NIGMS R01 692 GM083925（资助者ID：10.13039 / 100000057）的支持。
Materials
| Aluminum foil | Reynolds Group Holdings | Reynolds Wrap | Sterile tissue harvest |
| Aluminum-framed acrylic enclosure | Custom made | Friction tester component | |
| Autoclavable instant sealing sterilization pouches | Fisherbrand | 01-812-54 | Sterilization of tools |
| Autoclave | Buxton | Sterilization of tools | |
| Beaker (250 mL) | Pyrex Vista | 70000 | Tissue harvest |
| Betadine (Povidone Iodine Prep Solution) | Medline Industries, LP | MDS093906 | Sterile tissue harvest |
| Biological safety cabinet | Labconco | Purifier Logic+ Class II, Type A2 BSC | Sterile tissue harvest |
| Biospy punch | Steritool Inc. | 50162 | Tissue harvest |
| Box cutter | American Safety Razor Company | 94-120-71 | Tissue harvest |
| Circular acrylic-sillicone post (synovium) | Custom made | Tissue mounting | |
| Culture media | Custom made | DMEM (Cat No. 11-965-118; Gibco) supplemented with 50 μg/mL L-proline (Cat. No. P5607; Sigma), 100 μg/mL sodium pyruvate (Cat. No. S8636; Sigma), 1% ITS (Cat. No. 354350; Corning), and 1% antibiotic–antimycotic (Cat. No. 15-240-062, Gibco) | |
| Cyanoacrylate (Loctite 420 Clear) | Henkel | 135455 | Tissue mounting |
| Dead weights | OHAUS | Normal load | |
| Ethanol 200 proof | Decon Labs, Inc. | 2701 | Dilute to 70 % |
| Fixed base | ThorLabs, Inc. | SB1T | Friction tester component |
| Forceps (synovium harvest) | Fine Science Tools | 11019-12 | Tissue harvest |
| Forceps (synovium mounting) | Excelta | 3C-S-PI | Tissue mounting |
| Horizontal linear encoder (for translating stage) | RSF Electronics, Inc. | MSA 670.63 | Friction tester component; system resolution of 1 µm |
| Hot glue gun and glue | FPC Corporation | Surebonder Pro 4000A | Tissue mounting |
| LabVIEW | National Instruments Corporation | LabVIEW 2010 | Friction testing program |
| Load cell | JR3 Inc. | 20E12A-M25B | Friction tester component; 0.0019 lbs resolution in x&y, 0.0038 lbs resolution in z |
| Loading platen | Custom made | Tissue mounting | |
| O-ring | Parker | S1138AS568-009 | Tissue mounting |
| Petri dish (60 mm) | Falcon | 351007 | Tissue mounting |
| PivotLok Work Positioner (tibia holder) | Industry Depot, Pivot Lok | PL325 | Tissue harvest |
| Removable base | ThorLabs, Inc. | SB1B | Friction tester component |
| Ring stand | Tissue harvest | ||
| Scalpel blades | Havel's Inc. | FSC22 | Tissue harvest |
| Scalpel handle | FEATHER Safety Razor Co., Ltd. | No. 4 | Tissue harvest |
| Screwdriver | Wera | 3334 | Tissue harvest |
| Stage | JMAR | Friction tester component | |
| Stepper motor | Oriental Motor Co., Ltd. | PK266-03B | Friction tester component |
| Suction tool | Virtual Industries, Inc. | PEN-VAC Vacuum Pen | Tissue mounting |
| Support rod | Custom made | Tissue mounting | |
| Surgical scissors | Fine Science Tools | 14061-09 | Tissue mounting |
| Synovial fluid (bovine) | Animal Technologies, Inc. | Friction testing bath | |
| Testing bath | Custom made | Phosphate-Buffered Saline (PBS) with protease inhibitors: 0.04% isothiazolone-base biocide (Proclin 950 Cat. No. 46878-U; Sigma) and 0.1% protease inhibitor – 0.05 M ethylenediaminetetraacetic acid, EDTA (Cat. No. 0369; Sigma) | |
| Tissue culture incubator | Fisher Scientific | Isotemp | Sterile culture |
| Vertical linear encoder (for loading stage) | Renishaw | T1031-30A | Friction tester component; 20 nm resolution |
| Voice coil actuator | H2W Technologies | NCC20-15-027-1RC | Friction tester component |
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Cite This Article
Gangi, L. R., Petersen, C. A., Oungoulian, S. R., Estell, E. G., Durney, K. M., Suh, J. T., Ateshian, G. A., Hung, C. T. A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation. J. Vis. Exp. (184), e63880, doi:10.3791/63880 (2022).