JoVE Journal > Medicine
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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의학

一种可重复的软骨冲击模型在兔子中产生创伤后骨关节炎

Published: November 21, 2023
doi:
10.3791/64450
, , , , , , , , ,
1Department of Anatomy, Cell Biology and Physiology,Indiana University School of Medicine, 2Department of Orthopaedic Surgery,Indiana University School of Medicine, 3Indiana Center for Musculoskeletal Health,Indiana University School of Medicine, 4School of Mechanical Engineering,Purdue University, 5Large Animal Resource Center,Indiana University School of Medicine, 6Department of Mechanical and Energy Engineering,Indiana University-Purdue University Indianapolis

Summary

兔子开放的股内侧髁突冲击模型对于研究创伤后骨关节炎 （PTOA） 和缓解 PTOA 进展的新治疗策略是可靠的。该协议使用带有撞击头的基于托架的滴塔在兔子中产生后内侧股骨髁的孤立软骨缺损。

Abstract

创伤后骨关节炎 （PTOA） 占美国所有骨关节炎病例的 12%。PTOA 可由单个创伤事件引发，例如作用在关节软骨上的高冲击负荷，或由关节不稳定引发，如前交叉韧带断裂。目前尚无有效的治疗方法来预防 PTOA。开发可靠的 PTOA 动物模型对于更好地了解软骨损伤的机制以及研究缓解或预防 PTOA 进展的新治疗策略是必要的。该协议描述了一种开放的、基于落塔的兔股骨髁冲击模型，以诱导软骨损伤。该模型的峰值载荷为 579.1 ± 71.1 N，峰值应力为 81.9 ± 10.1 MPa，峰值载荷时间为 2.4 ± 0.5 ms。与未阻生的对侧 MFC（0.56 ± 0.42）和受阻膝的其他软骨表面相比，来自嵌塞内侧股骨髁 （MFC） 的关节软骨具有更高的凋亡细胞率 （p = 0.0058），并具有更高的国际骨关节炎研究学会 （OARSI） 评分 3.38 ± 1.43 （<0.56 0.42 ）。在未受影响的关节面中未检测到OARSI评分的差异（p > 0.05）。

Introduction

创伤后骨关节炎 （PTOA） 是全球致残的主要原因，占症状性骨关节炎 （OA） 的 12%-16%1。目前终末期 OA 管理的金标准是全膝关节和髋关节置换术2 或关节融合术，如终末期胫距关节或距下关节炎。虽然在很大程度上是成功的，但关节置换术可能会产生昂贵且病态的并发症3.此外，鉴于 77%-83%4,5 的无翻修种植体存活率较低，因此 50 岁以下的患者不太希望进行关节置换术。目前，尚无 FDA 批准的治疗方法来预防或减轻 PTOA 的进展。

PTOA 累及整个关节，包括滑膜组织、软骨下骨和关节软骨。其特征是关节软骨退化、滑膜炎症、软骨下骨重塑和骨赘形成 6,7。PTOA 的表型是通过软骨、滑膜和软骨下骨之间相互作用的复杂过程发展而来的。目前的理解是，软骨损伤导致细胞外基质 （ECM） 成分的释放，例如 2 型胶原 （COL2） 和聚集聚糖 （ACAN）。这些 ECM 组分片段具有促炎作用，可导致 IL-6、IL-1β 和活性氧的产生增加。这些介质作用于软骨细胞，导致基质金属蛋白酶 （MMP） 上调，例如 MMP-13，其降解关节软骨，同时也减少基质合成，导致关节软骨的整体分解代谢环境8。此外，有证据表明原发性骨关节炎和 PTOA 的软骨细胞凋亡增加 9,10。线粒体功能障碍发生在软骨11,12,13,14 的超生理负荷后，可导致软骨细胞凋亡增加 12,15。软骨细胞凋亡增强与蛋白多糖耗竭和软骨分解代谢增加有关，并已被证明先于软骨变化和软骨下骨重塑16,17,18。

与大多数人类疾病一样，需要可靠的 PTOA 转化模型来进一步了解疾病的病理生理学并测试新的治疗方法。猪和犬科动物等大型动物已被用于 PTOA17,19 的关节内骨折和冲击模型，但它们成本高昂。较小的动物模型，如小鼠、大鼠和兔子，成本较低，用于研究通过关节不稳定产生的 PTOA，这通常涉及前交叉韧带 （ACL） 的手术横断和/或内侧半月板的破坏 20、21、22、23、24、25。尽管关节创伤会导致各种后果，包括韧带损伤26，但几乎所有病例都会发生软骨的机械性超负荷。

越来越多的证据表明，韧带不稳定（如前交叉韧带横断）和急性软骨损伤后 PTOA 发展背后的病理学是由于不同的机制27。因此，开发软骨直接损伤模型非常重要。目前在大鼠和小鼠中产生骨软骨或软骨损伤的冲击模型数量有限28,29。然而，小鼠软骨不太适合产生孤立的软骨缺损。这是因为小鼠关节软骨只有 3-5 个细胞层厚，缺乏有组织的浅表、放射状和过渡软骨区，以及在人类和大型动物中发现的厚钙化软骨层。小鼠模型还显示部分软骨缺损的自发消退30,31。因此，我们选择了兔子作为这个冲击模型，因为它的软骨厚度和组织与人类相似，而且它是最小的动物模型，可以提供一致的软骨冲击，导致 PTOA。先前对兔子股骨髁撞击的开放手术模型采用了钟摆32、手持式弹簧加载软骨嵌塞装置 33 和允许创建兔子特异性撞击器的下降塔34。然而，这些研究缺乏体内数据。其他人已经报道了基于摆锤 35、气动36 和弹簧加载37 冲击装置10 的体内数据，这些研究表明，这些方法之间的峰值应力和加载速率的变异率很高。尽管如此，该领域仍缺乏一种一致的方法来可靠地模拟体内急性软骨创伤。

目前的方案采用基于落塔的系统，对兔膝关节的后内侧髁进行一致的冲击。采用膝关节后入路暴露股骨后内侧髁。然后将 Steinman 销从内侧到外侧穿过股骨髁，与关节面成一条直线，并固定在平台上。固定后，负荷被传递到股骨后内侧髁。这种方法允许将一致的软骨损伤传递到兔股骨远端的承重表面。

Protocol

以下程序是在印第安纳大学医学院机构动物护理和使用委员会 （IACUC） 的批准下执行的。所有生存手术均在无菌条件下进行，如美国国立卫生研究院指南所述。使用适当的镇痛药和抗生素来控制疼痛和感染风险，以优化成功的结果。本研究采用骨骼成熟的雄性新西兰白兔，体重3.0-4.0公斤。 1. 落塔制造 生成落塔、底座平台和机构组件的 CAD 图纸，以固定 St…

Representative Results

该手术的成功在撞击后立即通过外科医生对髁突的可视化（图4A）和射线照相来监测，以确保没有发生骨折（图4B）。存在冲击失败的风险，导致术中髁突骨折。这通常是由于 Steinman 引脚放置不当造成的（图 5）。使用该模型，继发于术中骨折的骨折失败率为 9.0%（67 例手术中的 6 例）。平均峰值冲击应力为81.9 ± 10.1 MPa（CV = 12.3%?…

Discussion

该外科手术旨在在 PTOA 模型中对兔股骨内侧髁的承重表面产生一致的软骨损伤。该手术的一个优点是膝关节的后入路允许直接观察完整的后股内侧髁，并且可以在大约 37 分钟内完成（表 2）。还应该注意的是，这是一个开放性损伤模型，除了由于滑膜和关节囊的潜在损伤而导致的影响之外，还可能导致急性炎症变化17,44。表3总?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项研究得到了美国陆军医学研究收购活动的 DoD 同行评审医学研究计划 – 研究者发起的研究奖 W81XWH-20-1-0304、NIH NIAMS R01AR076477和 NIH （AR065971） 的综合肌肉骨骼 T32 培训计划以及 NIH NIAMS Grant R01 AR069657 的支持。作者要感谢 Kevin Carr 为该项目提供他在加工和制造方面的专业知识，并感谢 Drew Brown 和印第安纳州肌肉骨骼健康骨骼组织学中心对组织学的帮助。

Materials

Flat head screw McMaster-Carr 92210A194 Stainless steel hex drive flat head screw, 8-32, 1/2"
#15 scalpel blades McKesson 1029066 Scalpel McKesson No. 15 Stainless Steel / Plastic Classic Grip Handle Sterile Disposable
1/2”-20 threaded rod McMaster-Carr 99065A120 1/2”-20 threaded rod
10 mL syringe McKesson 1031801 For irrigation; General Purpose Syringe McKesson 10 mL Blister Pack Luer Lock Tip Without Safety
3 mL syringe McKesson 1031804 For lidocaine/bupiviacaine injection; General Purpose Syringe McKesson 3 mL Blister Pack Luer Lock Tip Without Safety.
3-0 polysorb Ethicon J332H 3-0 Vircryl, CT-2, 1/2 circle, 26 mm, tapered
4-0 monosorb Ethicon Z397H 4-0 PDS 2, FS-2, 3/8 circle, 19mm, cutting edge
5-0 polysorb Med Vet International NC9335902 Med Vet International 5-0 ETHICON COATED VICRYL C-3
Accelerometer Kistler 8743A5 Accelerometer
Adson-Browns Forceps World precision tools 500177 Adson-Brown Forceps, 12 cm, Straight, TC Jaws, 7 x 7 Teeth
Alfaxalone Jurox 49480-002-01 Alfaxan Multidose by Jurox : 10 mg/mL
Buprenorphine Par Pharmaceuticals 42023-0179-05 Buprenorphine HCL injection: 0.3 mg/mL
Butorphanol  Zoetis 54771-2033 Butorphanol tartrate 10mg/ml by Zoetis
Chlorhexidine Hand Scrub BD 371073 BD E-Z Scrub 107 Surgical Scrub Brush/Sponge, 4% CHG, Red
Collet STRYKER 14023 Stryker 4100-62 wire Collet 0.28-0.71''
Cordless Driver handpiece STRYKER OR-S4300 Stryker 4300 CD3 Cordless Driver 3 handpiece
Cricket Retractors Novosurgical G3510 21 2x Heiss (Holzheimer) Cross Action Retractor
Dissector Scissors Jorvet labs J0662 Aesculap AG, Metzenbaum, Scissors, Straight 5 3/4″
Elizabethian Collar ElizaSoft 62054 ElizaSoft Elizabethan Recovery Collar
Enrofloxacin Custom Meds Enrofloxacin compounded by Custom Meds
Eye Ointment Pivetal  46066-753-55 Pivetal Articifical Tears- recently recalled
Face-mount shaft collar McMaster-Carr 5631T11 Face-mount shaft collar
Fast green Millipore Sigma F7258 Fast green
Freer Jorvet labs J0226Q Freer elevator
Head screw -1 McMaster-Carr 91251A197 Black-oxide alloy steel socket head screw, 8-32, 3/4"
Head screw -2 McMaster-Carr 92196A194 Stainless steel socket head screw, 8-32, 1/2"
Head screw -3 McMaster-Carr 92196A146 Stainless steel socket head screw, 8-32, 1/2"
Head screw -4 McMaster-Carr 92196A151 Stainless steel socket head screw, 6-32, 3/4"
Hematoxylin Solution, Gill No. 1 Millipore Sigma GHS132-1L Hematoxylin Solution, Gill No. 1
Hex nut McMaster-Carr 91841A007 Stainless steel hex nut, 6-32
Hold-down toggle clamp McMaster-Carr 5126A71 Hold-down toggle clamp
Impact device n/a n/a custom made
Impact platform n/a n/a custom made
K-wires Jorvet Labs J0250A JorVet Intramedullary Steinman Pins, Trocar-Trocar 1/16" x 7"
Lab View National Instruments n/a n/a
Load cell Kistler 9712B5000 Load cell
MATLAB The MathWorks Inc. n/a n/a
Microscope Leica DMi-8 Leica DMi8 microscope with LAS-X software
Midazolam Almaject 72611-749-10 Midazolam Hydrochloride injection: 5mg/ml by Almaject
milling machine depth stops McMaster-Carr 2949A71 Clamp-on milling machine depth stops
Mobile C-arm Philips 718095 BV Pulsera, Mobile C-arm
Mounted linear ball bearing McMaster-Carr 9338T7 Mounted linear ball bearing
Needle Driver A2Z Scilab A2ZTCIN39 TC Webster Needle Holder Smooth Jaws 5", Premium
Pentobarbital Vortech 0298-9373-68 Pentobarbital 390 mg/mL by Vortech
Safranin O Millipore Sigma HT90432 Safranin O
Small Battery pack STRYKER NS014036 6212 Small Battery pack- 9.6 V
Steel rod, 2’ McMaster-Carr 89535K25 Steel rod, 2’
Sterile Saline ICU Medical 6139-22 AquaLite Solution Pour Bottles, 250 mL
Stryker 6110-120 System 6 Battery Charger STRYKER OR-S6110-120
Surgical gloves McKesson 1044729 Surgical Glove McKesson Perry Size 6.5 Sterile Pair Latex Extended Cuff Length Smooth Brown Not Chemo Approved
Surgical gown McKesson 1104452 Non-Reinforced Surgical Gown with Towel McKesson Large Blue Sterile AAMI Level 3 Disposable
Suture scissors Jorvet Labs J0910SA Super Cut Scissors, Mayo, Straight, 5 1/2″
TUNEL staining kit ABP Bioscience A049 TUNEL Chromogenic Apoptosis Detection Kit
Weitlaner Retractors Fine Science Tools 17012-11 2x Weitlaner-Locktite Retractors
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Tags

Cartilage Impact ModelPost-traumatic OsteoarthritisRabbitSteinmann PinFemoral ShaftArthrotomyMedial Femoral CondyleJoint TraumaTherapeutic Evaluation
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Dilley, J., Noori-Dokht, H., Seetharam, A., Bello, M., Nanavaty, A., Natoli, R. M., McKinley, T., Bault, Z., Wagner, D., Sankar, U. A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit. J. Vis. Exp. (201), e64450, doi:10.3791/64450 (2023).
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