植入物关节软骨滑向金属的生物摩擦学测试与分析
Summary
该协议描述了骨质体油柱在金属植入物材料上滑动的制备、生物摩擦测试和分析。该协议中包含的结果指标是代谢活性、基因表达和组织学。
Abstract
中年患者的骨质疏松缺陷可能用焦点金属植入物进行治疗。首先开发为膝关节的缺陷，植入物现在可用于肩部，臀部，脚踝和第一个骨质性关节。在提供疼痛减轻和临床改善的同时，许多患者观察到对立软骨的渐进性退行性变化。导致这种损害的机制尚未完全了解。该协议描述了一个摩擦实验，以模拟软骨上的金属配对和关节软骨的综合分析。金属植入物材料测试牛骨柱作为人体关节软骨的模型。通过应用不同的负载和滑动速度，可以模拟生理载荷条件。为了全面分析关节软骨的影响，本协议描述了组织学、代谢活性和基因表达分析。摩擦学测试的主要优点是，可以自由调节载荷参数，以模拟体内条件。此外，不同的测试解决方案可用于研究润滑或亲炎剂的影响。通过使用软骨特异性基因和猫科动物基因的基因表达分析，可以检测到关节软骨细胞代谢的早期变化，以回应机械负荷。
Introduction
骨质疏松缺陷的治疗要求很高，在许多情况下需要手术。对于中年患者的聚焦骨质病变，焦点金属植入物是一个可行的选择，尤其是在原发治疗失败之后，如骨髓刺激（BMS）或自体软骨细胞植入（ACI）1。部分表面置换可视为挽救程序，可减轻疼痛，改善运动范围2。这些植入物通常由CoCrMo合金组成，具有不同的尺寸和偏移配置，以匹配正常的解剖结构3。虽然最初开发为膝盖的内侧股骨孔的缺陷，这种植入物现在可用，并用于臀部，脚踝，肩膀和肘部4，5，6。为了获得满意的结果，评估相对软骨的机械关节对齐和状况至关重要。此外，正确的植入没有突起植入物已被证明是基本的7。
临床研究显示，在减少疼痛及改善中年病人功能方面，有优良的短期效果。与同体植入相比，焦金属植入物允许早期承重。然而，对立的关节软骨显示加速磨损在相当多的病人9，10。因此，即使放置适当，在许多情况下，原生软骨的退化似乎也不可避免，而基本机制仍然不清楚。类似的退行性变化已经观察到后，双相体体成形术的臀部11和增加与活动和加载12。
摩擦实验提供了在体外研究这种配对和模拟生理条件下发生的不同载荷情况的可能性。骨质针的使用提供了一个简单的几何模型，以研究关节软骨滑对原生软骨或任何植入材料14的摩擦学， 并可以进一步用于整个关节模拟模型15。软骨上的金属配对显示软骨磨损加速，细胞外基质中断，和减少细胞生存能力在表面区域相比软骨对软骨配对16。软骨的损伤主要发生在浅层和中间区域17之间的分层形式。然而，导致软骨退化的机制尚未完全了解。该协议对关节软骨的生物合成活性进行了综合分析。通过确定代谢活性和代谢基因的基因表达水平，可以确定软骨分解的早期适应症。体外摩擦学实验的优点是可以调整载荷参数以模拟各种载荷条件。
因此，以下协议适合模拟软骨上的金属配对，代表实验性半人成形模型。
Protocol
1. 金属钢瓶的准备 分析圆柱形钴-铬-硅（CoCrMo）棒，使用扫描电子显微镜（SEM）和每个制造商的协议能量分散X射线光谱，以确认所提供的值，符合手术植入物的化学成分标准规范。注：用于此实验的CoCrMo合金元素成分为65%Co、28%Cr、5%Mo和2%其他。 用碳化硅研磨纸湿磨样品，从粒度为 500 开始。使用研磨纸，增加顺序高达 4000 的颗粒大小。 用 3 μm 和 1 μm 糊进行抛光，以达?…
Representative Results
必须使用压力测量膜确认接触区域和接触压力（图1）。生理载荷条件可以通过与所定义的接触压力的参考印迹进行比较来确认。在测试过程中，摩擦系数不断被监测。对于迁移接触区，低摩擦系数可保持至少 1 小时（图 2）。使用萨夫兰宁O染色的细胞外基质组成和结构可以确定（图3）。萨夫拉宁 O 染色的强度与蛋白细胞素含量?…
Discussion
聚焦金属植入物是骨质疏松缺陷的抢救程序，特别是在中年患者和初级治疗失败后。虽然临床研究表明有希望的短期结果，一个观察到的并发症是损害对立的，本地软骨10。尸体和生物力学研究表明，适当的植入与平坦或轻微凹陷定位保持自然接触压力19。摩擦实验提供了在体外测试各种软骨配对的可能性。在这种情况下，装载条件、润滑、材料配对和持续时间…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项研究由Né Forschungs-und Bildungsges.m.b.H.和下奥地利省政府通过生命科学电话（项目ID：LSC15-019）和奥地利彗星方案（项目K2 XTribology，第849109号赠款）。
Materials
| Amphotericin B | Sigma‐Aldrich Chemie GmbH | A-2942-100ML | |
| buffered formaldehyde solution 4% | VWR | 97131000 | |
| Cell Proliferation Kit II (XTT) | Roche Diagnostics | 11465015001 | XTT-based ex vivo toxicology assay |
| CoCrMo raw material | Acnis International | CoCrMo rods 6mm in diameter | |
| CryoStar NX70 Cryostat | Thermo Fischer Scientific | cryosectioning device | |
| dimethyl sulfoxide (DMSO) | Sidma-Aldrich Chemie | D 2438-10ML | |
| Dulbecco’s modified Eagle’s medium | Sigma‐Aldrich Chemie GmbH | medium | |
| fetal bovine serum | Gibco | ||
| Hyaluronic acid | Anika Therapeutics Inc. | component of lubricating solution | |
| iCycler | BioRad | thermal cycler | |
| Leica microscope DM‐1000 | Leica | microscope for histology | |
| LightCycler 480 Sealing Foil | Roche Diagnostics | ||
| LightCycler 96 | Roche Diagnostics | thermal cycler for PCR | |
| MagNA Lyser Green Beads | Roche Diagnostics | 3358941001 | |
| Osteochondral Autograft Transfer System (OATS) | Arthrex Inc. | cutting tube for harvesting osteochondral cylinders | |
| osteosoft | Merck | 1017279010 | decalcifier-solution |
| Penicillin /Streptomycin | Sigma‐Aldrich Chemie GmbH | P4333-100ML | |
| phosphate‐buffered saline | Sigma‐Aldrich Chemie GmbH | PBS | |
| Prescale Low Pressure | Fujifilm | pressure indicating film | |
| RNeasy Fibrous Tissue Kit | QIAGEN | 74404 | |
| Synergy 2 | BioTek Instruments | plate reader | |
| Tetra‐Falex MUST | Falex Tribology | Tribometer | |
| Tissue‐ Tek O.C.T. | SAKURA | 4583 | embedding formulation |
| Transcriptor First Strand cDNA Synthesis Kit | Roche Diagnostics | 40897030001 | |
| β-mercaptoethanol | Sidma-Aldrich Chemie | M3148 |
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Stotter, C., Bauer, C., Simlinger, B., Ripoll, M. R., Franek, F., Klestil, T., Nehrer, S. Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants. J. Vis. Exp. (159), e61304, doi:10.3791/61304 (2020).