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Bio-ingénierie

小鼠后肢外植体研究跟腱撞击力学生物学的模型

Published: December 08, 2023
doi:
10.3791/65801
, , ,
1Department of Biomedical Engineering,University of Rochester, 2Department of Orthopaedics, Center for Musculoskeletal Research,University of Rochester Medical Center, 3Department of Pharmacology and Physiology,University of Rochester Medical Center

Summary

我们提出了一个定制的实验平台和组织培养方案，该方案以持续的细胞活力再现了由小鼠后肢外植体中跟腱插入撞击驱动的纤维软骨变化，提供了一个适合探索肌腱撞击的机制生物学的模型。

Abstract

肌腱撞击骨骼会产生多轴机械应变环境，横向压缩应变明显升高，从而引发局部纤维软骨表型，其特征是富含糖胺聚糖 （GAG） 的基质积累和胶原网络的重塑。虽然纤维软骨是健康肌腱撞击区域的正常特征，但过多的 GAG 沉积和胶原网络紊乱是肌腱病的标志性特征。因此，撞击在临床上被认为是肌腱病发生和发展的重要外在因素。然而，肌腱撞击背后的机制生物学研究仍未得到充分研究。先前阐明细胞对肌腱撞击反应的努力已经对细胞施加了单轴压缩，并在 体外切除了肌腱外植体。然而，分离的细胞缺乏对机械反应至关重要的三维细胞外环境，并且 体外 和切除的外植体研究都无法概括 体内肌腱撞击产生的多轴应变环境，这取决于撞击区域的解剖学特征。此外， 肌 腱撞击的体内模型缺乏对机械应变环境的控制。为了克服这些局限性，我们提出了一种适用于研究跟腱撞击机制生物学的新型小鼠后肢外植体模型。该模型将跟腱保持在 原位 以保持局部解剖结构，并再现了在被动应用踝关节背屈期间跟腱插入跟骨上产生的多轴应变环境，同时将细胞保留在其原生环境中。我们描述了该模型不可或缺的组织培养方案，并提供了在 7 天内建立持续外植体活力的数据。代表性结果表明，组织学 GAG 染色增强，继发于撞击的胶原纤维排列减少，表明纤维软骨形成增加。该模型可以很容易地适应研究不同的机械负荷方案，并允许操纵感兴趣的分子途径，以确定介导跟腱表型变化响应撞击的机制。

Introduction

许多肌腱，包括跟腱和肩袖肌腱，由于正常的解剖定位而经历骨撞击1,2,3,4.肌腱撞击产生横向指向纵向纤维轴的压缩应变5,6,7.肌腱撞击区域表现出独特的纤维软骨表型，其中收缩的圆形细胞（纤维软骨细胞）嵌入无组织的胶原网络中，糖胺聚糖 （GAG） 含量显着增加2,3,4,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24.先前的研究表明，肌腱撞击产生的不同机械环境通过驱动大型聚集蛋白聚糖（尤其是聚集聚糖）的沉积来维持这种富含GAG的基质，尽管其潜在机制尚不清楚1,3,12,13,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39.虽然纤维软骨是健康肌腱撞击区域的正常特征，但与纤维软骨形成过度相关的异常蛋白多糖代谢是肌腱病的标志特征，肌腱病是一种常见且使人衰弱的疾病，不成比例地出现在慢性撞击肌腱中1,40,41,42,43,44,45,46,47,48,49.因此，肌腱撞击在临床上被认为是驱动几种最常见的肌腱病的重要外在因素，包括肩袖疾病和插入性跟腱病 （IAT）50,51,52.目前，肌腱病的治疗效率低下。例如，大约 47% 的 IAT 患者在保守治疗失败后需要手术干预，术后结局各不相同53,54,55,56.尽管撞击和肌腱病之间存在明显的关系，但撞击肌腱细胞感知和响应其机械环境的机械生物学机制描述甚少，这模糊了对肌腱病发病机制的理解并导致治疗不充分。

外植体模型是肌腱机械生物学研究的有用工具57,58。作为了解肌腱撞击机制生物学的第一步，一些先前的研究已经探索了对细胞或切除的肌腱外植体应用简单单轴压缩后的细胞反应 27,29,30,31,32,33,34,39。然而，体外细胞缺乏细胞外和细胞周基质，这些基质可促进菌株转移，隔离机械变形释放的重要生长因子和细胞因子，并为在机械转导中发挥作用的粘着斑复合物提供底物57,59。此外，体外和切除的外植体研究都无法概括体内肌腱撞击产生的多轴机械应变环境，这取决于撞击区域的解剖学特征 5,6。在跟腱插入受撞击的情况下，这包括周围组织，例如跟骨后滑囊和 Kager 的脂肪垫 60,61,62,63。相反，肌腱撞击的体内模型 25,28,36,37,38,64,65,66 允许对直接施加到肌腱的载荷的大小和频率进行最小的控制，这是研究肌腱机械生物学的体内模型的一个公认的局限性 57,58，67,68,69,70.鉴于在体内测量肌腱应变的挑战，这些模型中产生的内部应变环境通常表征不佳。

在这篇手稿中，我们提出了一个定制的实验平台，该平台在整个小鼠后肢外植体中重建了跟骨上的跟腱插入的冲击，当与这种组织培养方案配对时，在外植体培养中保持活力超过 7 天，并允许研究肌腱撞击的生物学后遗症。该平台建立在 3D 打印的聚乳酸 （PLA） 基座上，为夹具和 3D 打印 PLA 减容插件的连接奠定了基础。握把用于将大腿和膝盖夹紧在跟腱肌腱交界处的近端，后肢的尾侧朝上，允许使用超声探头或倒置显微镜从上方对跟腱进行成像（图1A）。减容插入物沿着底座上的轨道滑动，并减少所需的组织培养基体积。缠绕在后爪上的编织线利用基础设计和 3D 打印的 PLA 夹从平台中布线。通过拉动绳子，后爪背屈，跟腱插入物撞击跟骨，导致横向压缩应变升高 5,6（图 1A）。该平台包含在丙烯酸浴中，该浴将后肢外植体浸没在组织培养基中。用胶带将绷紧的绳子固定在浴缸的外面，可以保持脚踝背屈，从而对跟腱插入产生静电冲击。3D 打印组件的 CAD 文件以多种格式提供（补充文件 1），允许导入到一系列商业和免费的开源 CAD 软件中进行修改以满足实验需求。如果无法使用 3D 打印机进行制造，则可以将 CAD 文件提供给在线 3D 打印服务，这些服务将以低成本打印和运输零件。

重要的是，肱三头肌-跟腱肌腱复合体横跨膝关节和踝关节 71,72,73。因此，跟腱的拉伸拉伤受膝关节屈曲的影响。膝关节伸展使跟腱处于紧张状态，而膝关节屈曲可降低张力。通过首先伸展膝盖，然后被动地背屈踝关节，撞击插入处的压缩应变可以叠加在拉伸应变上。相反，通过在膝关节屈曲的情况下被动地背屈踝关节，拉伸应变会减少，并且压缩应变仍然存在。目前的协议探讨了三种这样的条件。1） 对于静态撞击，足背弯曲相对于胫骨 < 110° 以撞击插入物，膝盖弯曲以减少张力。2） 对于基线张力组，踝关节伸展至背屈 145° 以上，膝关节伸展，在插入处产生主要拉伸应变。3）对于卸载组，在没有外部施加负荷的情况下，将外植体培养在培养皿中，膝盖和脚踝处于中立位置。上述角度是相对于坐标系进行照相测量的，其中足部和胫骨以 180° 的角度平行，以 90° 的角度垂直。

该方案的关键步骤包括 1） 解剖后肢外植体并小心切除皮肤和足底肌腱;2）地塞米松预处理48小时后的外植体培养;3）组织切片和组织学染色;4）彩色图像分析以评估纤维软骨的形成。解剖后，将每个后肢外植体在补充有地塞米松74的培养基中预处理48小时。将每只小鼠的对侧肢体分配到单独的实验组进行成对比较，这有助于控制生物学变异性。预处理后，将外植体如上所述放置在平台中并再培养7天（图1B）。与预处理组（第 0 天）进行额外的比较，其中在预处理 48 小时后立即去除外植体。

外植体培养后，修剪后肢，将福尔马林固定，脱钙并包埋在石蜡中。矢状面方向的连续切片提供了从肌腱交界处到跟骨插入处的跟腱的可视化，同时允许通过整个肌腱跟踪切片深度。末端脱氧核苷酸转移酶 （TdT） 介导的 dUTP X-nick 标记 （TUNEL） 用于可视化继发于细胞凋亡的 DNA 损伤并评估活力。进行甲苯胺蓝组织学和定制彩色图像分析以量化 GAG 染色的变化。然后将甲苯胺蓝染色的组织切片用于SHG成像，以表征胶原纤维组织的改变（图1B）。

提供的代表性结果表明，富含GAG的基质的组织学染色改变，模型内7天的静态撞击产生的细胞外胶原网络紊乱。该模型可用于探索撞击驱动的纤维软骨变化的分子机制。

Protocol

所有动物工作均由罗切斯特大学动物资源委员会批准。 1.组织培养基的制备 在37°C和5%CO2的培养箱中，在Dulbecco改良的Eagle培养基（1x DMEM）中培养所有外植体，其中含有1%v / v青霉素 – 链霉素和200μM L-抗坏血酸。对于最初的 48 小时预处理，在补充有 100 nM 地塞米松74 的 70 mL 培养基中培养每个外植体。预处理后，在没有地塞米松的情?…

Representative Results

TUNEL染色组织切片的代表性图像显示，在实验组的外植体培养7天后，跟腱体内的凋亡核最小（图2A）。这些图像的定量提供了证据，证明在外植体培养 7 天后，组织培养方案在不同负载条件下的外植体培养 7 天后，在跟腱内平均保持高达 78% 的活力（图 2B）。 定性地，与未加载的对照组相比，在静电冲击 7 天后，增强的甲苯胺蓝染?…

Discussion

实验性小鼠后肢外植体平台与本研究中描述的组织培养方案相结合，为研究跟腱插入处撞击驱动的纤维软骨形成的机制生物学提供了合适的模型。代表性结果证明了该外植体模型的实用性，该结果表明，在静态撞击 7 天后，甲苯胺蓝染色的显着和空间异质性变化同时维持了细胞活力。这些发现表明继发于机械撞击的富含GAG的基质分子的代谢改变，与其他模型和临床研究一致</…

Divulgations

The authors have nothing to disclose.

Acknowledgements

作者感谢罗切斯特大学肌肉骨骼研究中心组织学、生物化学和分子成像 （HBMI） 核心的 Jeff Fox 和 Vidya Venkatramani 提供的支持和帮助，部分资金来自 P30AR06965。此外，作者要感谢罗切斯特大学医学中心的光学显微镜和纳米镜中心（CALMN）在多光子显微镜方面的帮助。这项研究由 R01 AR070765 和 R01 AR070765-04S1 以及 1R35GM147054 和 1R01AR082349 资助。

Materials

Absorbent underpads VWR 82020-845 For benchtop dissection
Acrylic bath Source One X001G46CB1 Contains the explant platform submerged in culture media
Autoclave bin Thermo Scientific 13-361-20 Used as secondary containment, holds two platforms
Base – – 3D printed from CAD files provided as Supplementary Files
Braided line KastKing 30lb test Used to wrap around paw and apply ankle dorsiflexion
Clip – – 3D printed from CAD files provided as Supplementary Files
Cover glass Fisherbrand 12-541-034 Rectangular, No. 2, 50 mm x 24 mm
Cytoseal XYL VWR 8312-4 Xylene-based mounting media for coverslipping Toluidine blue stained tissue sections
Dexamethasone MP Biomedical LLC 194561 CAS#50-02-2
Dimethyl sulfoxide (DMSO), anhydrous Invitrogen by ThermoFisher D12345 CAS#67-68-5, use to solubilize dexamethasone into concentrated stock solutions
Double-sided tape Scotch Brand 34-8724-5195-9 To attach sandpaper to Grip platens
Dulbecco's Modified Eagle Medium (1X DMEM) Gibco by ThermoFisher 11965092 high glucose, (-) pyruvate, (+) glutamine
EDTA tetrasodium salt dihydrate Thermo Scientific Chemicals J15700.A1 CAS#10378-23-1, used to make 14% EDTA solution for sample decalcifcation
Ethanol, 200 proof Thermo Scientific T038181000 CAS#64-17-5, 1 L supply
Foam biopsy pads Leica 3801000 Used with processing cassettes, help hold ankle joints in desired position during fixation and decalcification
Forceps, #SS Standard Inox Dumont 11203-23 Straight, smooth, fine tips
Forceps, Micro-Adson 4.75" Fisherbrand 13-820-073 Straight, fine tips with serrated teeth
Garnet Sandpaper, 50-D Grit Norton M600060 01518 Or other coarse grit sandpaper
Glacial acetic acid Fisher Chemical A38S-500 CAS#64-19-7, for adjusting pH of sodium acetate buffer used for Toluidine blue histology, as well as 14% EDTA decalcification solution
Grips ADMET GV-100NT-A4 Stainless steel vice grips, screws and springs described in the protocol are included
Histobond Adhesive Microscope Slides VWR 16005-108 Sagittal sections of hind limbs explants reliably adhere to these slides through all staining protocols
In situ Cell Death Detection Kit, TMR Red Roche 12156792910 TUNEL assay
Labeling tape Fisherbrand 15-959 Or any other labeling tape of preference
L-ascorbic acid Sigma-Aldrich A4544-100G CAS#50-81-7, for culture media formulation
Neutral buffered formalin, 10% Leica 3800600 For sample fixation, 5 gallon supply
Nunc petri dishes Sigma-Aldrich P7741-1CS 100 mm diameter x 25 mm height, maintain explants submerged in 70 mL of culture media as described in protocol
Penicillin-streptomycin (100X) Gibco by ThermoFisher 15140122 Add 5 mL to 500 mL 1X DMEM for 1% v/v (1X) working concentration
Polylactic acid (PLA) 1.75 mm filament Hatchbox – Choose filament diameter compatible with your 3D printer extruder, in color of choice.
Processing cassettes Leica 3802631 For fixation, decalcification and paraffin embedding
Prolong Gold Antifade Reagent with DAPI Invitrogen by ThermoFisher P36931 Mounting media for coverslipping tissue sections after TUNEL
Proteinase K Fisher BioReagents BP1700-50 CAS#39450-01-6, used for antigen retrieval in TUNEL protocol
Scissors, Fine FST 14094-11 Straight, sharp
Slide Staining Set, 12-place Mercedes Scientific  MER 1011 Rack with 12 stain dishes and slide dippers for Toluidine blue histology
Sodium acetate, anhydrous Thermo Scientific Chemicals A1318430 CAS#127-09-3, used to make buffer for Toluidine blue histology
Tissue-Tek Accu-Edge Low Profile Microtome Blades VWR 25608-964 For paraffin sectioning
Toluidine Blue O Thermo Scientific Chemicals 348601000 CAS#92-31-9
Volume Reduction Insert – – 3D printed from CAD files provided as Supplementary Files
Xylenes Leica 3803665 4 gallon supply for histological staining
DOWNLOAD MATERIALS LIST

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Keywords: Murine Hind LimbExplant ModelMechanobiologyAchilles Tendon ImpingementMultiaxial Mechanical StrainFibrocartilageTendinopathyIn Vitro ModelIn Vivo ModelExtracellular MatrixGlycosaminoglycan (GAG)Collagen Network
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Wise, B. C., Mora, K. E., Lee, W., Buckley, M. R. Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement. J. Vis. Exp. (202), e65801, doi:10.3791/65801 (2023).
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