Name: 作者聚焦：揭开肌腱撞击的力学生物学 – 多轴鼠后肢外植体模型
Uploaded: 2023-12-08T13:30:00
Description: Watch this Scientific Journal Video about Unraveling the Mechanobiology of Tendon Impingement – A Multiaxial Murine Hind Limb Explant Model at JoVE.com

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。

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
<ol><li>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?...

了解肌腱撞击 – 用于研究机械生物学的小鼠外植体模型

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</...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Absorbent underpads</td>
			<td>VWR</td>
			<td>82020-845</td>
			<td>For benchtop dissection</td>
		</tr>
		<tr>
			<td>Acrylic bath</td>
			<td>Source One</td>
			<td>X001G46CB1</td>
			<td>Contains the explant platform submerged in culture media</td>
		</tr>
		<tr>
			<td>Autoclave bin</td>
			<td>Thermo Scientific</td>
			<td>13-361-20</td>
			<td>Used as secondary containment, holds two platforms</td>
		</tr>
		<tr>
			<td>Base</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Braided line</td>
			<td>KastKing</td>
			<td>30lb test</td>
			<td>Used to wrap around paw and apply ankle dorsiflexion</td>
		</tr>
		<tr>
			<td>Clip</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Fisherbrand</td>
			<td>12-541-034</td>
			<td>Rectangular, No. 2, 50 mm x 24 mm</td>
		</tr>
		<tr>
			<td>Cytoseal XYL</td>
			<td>VWR</td>
			<td>8312-4</td>
			<td>Xylene-based mounting media for coverslipping Toluidine blue stained tissue sections</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>MP Biomedical LLC</td>
			<td>194561</td>
			<td>CAS#50-02-2</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), anhydrous</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>D12345</td>
			<td>CAS#67-68-5, use to solubilize dexamethasone into concentrated stock solutions</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch Brand</td>
			<td>34-8724-5195-9</td>
			<td>To attach sandpaper to Grip platens</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (1X DMEM)</td>
			<td>Gibco by ThermoFisher</td>
			<td>11965092</td>
			<td>high glucose, (-) pyruvate, (+) glutamine</td>
		</tr>
		<tr>
			<td>EDTA tetrasodium salt dihydrate</td>
			<td>Thermo Scientific Chemicals</td>
			<td>J15700.A1</td>
			<td>CAS#10378-23-1, used to make 14% EDTA solution for sample decalcifcation</td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Thermo Scientific</td>
			<td>T038181000</td>
			<td>CAS#64-17-5, 1 L supply</td>
		</tr>
		<tr>
			<td>Foam biopsy pads</td>
			<td>Leica</td>
			<td>3801000</td>
			<td>Used with processing cassettes, help hold ankle joints in desired position during fixation and decalcification</td>
		</tr>
		<tr>
			<td>Forceps, #SS Standard Inox</td>
			<td>Dumont</td>
			<td>11203-23</td>
			<td>Straight, smooth, fine tips</td>
		</tr>
		<tr>
			<td>Forceps, Micro-Adson 4.75&#34;</td>
			<td>Fisherbrand</td>
			<td>13-820-073</td>
			<td>Straight, fine tips with serrated teeth</td>
		</tr>
		<tr>
			<td>Garnet Sandpaper, 50-D Grit</td>
			<td>Norton</td>
			<td>M600060 01518</td>
			<td>Or other coarse grit sandpaper</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Chemical</td>
			<td>A38S-500</td>
			<td>CAS#64-19-7, for adjusting pH of sodium acetate buffer used for Toluidine blue histology, as well as 14% EDTA decalcification solution</td>
		</tr>
		<tr>
			<td>Grips</td>
			<td>ADMET</td>
			<td>GV-100NT-A4</td>
			<td>Stainless steel vice grips, screws and springs described in the protocol are included</td>
		</tr>
		<tr>
			<td>Histobond Adhesive Microscope Slides</td>
			<td>VWR</td>
			<td>16005-108</td>
			<td>Sagittal sections of hind limbs explants reliably adhere to these slides through all staining protocols</td>
		</tr>
		<tr>
			<td>In situ Cell Death Detection Kit, TMR Red</td>
			<td>Roche</td>
			<td>12156792910</td>
			<td>TUNEL assay</td>
		</tr>
		<tr>
			<td>Labeling tape</td>
			<td>Fisherbrand</td>
			<td>15-959</td>
			<td>Or any other labeling tape of preference</td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4544-100G</td>
			<td>CAS#50-81-7, for culture media formulation</td>
		</tr>
		<tr>
			<td>Neutral buffered formalin, 10%</td>
			<td>Leica</td>
			<td>3800600</td>
			<td>For sample fixation, 5 gallon supply</td>
		</tr>
		<tr>
			<td>Nunc petri dishes</td>
			<td>Sigma-Aldrich</td>
			<td>P7741-1CS</td>
			<td>100 mm diameter x 25 mm height, maintain explants submerged in 70 mL of culture media as described in protocol</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100X)</td>
			<td>Gibco by ThermoFisher</td>
			<td>15140122</td>
			<td>Add 5 mL to 500 mL 1X DMEM for 1% v/v (1X) working concentration</td>
		</tr>
		<tr>
			<td>Polylactic acid (PLA) 1.75 mm filament</td>
			<td>Hatchbox</td>
			<td>-</td>
			<td>Choose filament diameter compatible with your 3D printer extruder, in color of choice.</td>
		</tr>
		<tr>
			<td>Processing cassettes</td>
			<td>Leica</td>
			<td>3802631</td>
			<td>For fixation, decalcification and paraffin embedding</td>
		</tr>
		<tr>
			<td>Prolong Gold Antifade Reagent with DAPI</td>
			<td>Invitrogen by ThermoFisher</td>
			<td>P36931</td>
			<td>Mounting media for coverslipping tissue sections after TUNEL</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Fisher BioReagents</td>
			<td>BP1700-50</td>
			<td>CAS#39450-01-6, used for antigen retrieval in TUNEL protocol</td>
		</tr>
		<tr>
			<td>Scissors, Fine</td>
			<td>FST</td>
			<td>14094-11</td>
			<td>Straight, sharp</td>
		</tr>
		<tr>
			<td>Slide Staining Set, 12-place</td>
			<td>Mercedes Scientific&#160;</td>
			<td>MER 1011</td>
			<td>Rack with 12 stain dishes and slide dippers for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Sodium acetate, anhydrous</td>
			<td>Thermo Scientific Chemicals</td>
			<td>A1318430</td>
			<td>CAS#127-09-3, used to make buffer for Toluidine blue histology</td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Low Profile Microtome Blades</td>
			<td>VWR</td>
			<td>25608-964</td>
			<td>For paraffin sectioning</td>
		</tr>
		<tr>
			<td>Toluidine Blue O</td>
			<td>Thermo Scientific Chemicals</td>
			<td>348601000</td>
			<td>CAS#92-31-9</td>
		</tr>
		<tr>
			<td>Volume Reduction Insert</td>
			<td>-</td>
			<td>-</td>
			<td>3D printed from CAD files provided as Supplementary Files</td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Leica</td>
			<td>3803665</td>
			<td>4 gallon supply for histological staining</td>
		</tr>
	</tbody>
</table>

murine hind limb explant model for studying the mechanobiology of achilles tendon impingement

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。

A multitude of tendons, including the Achilles tendon and rotator cuff tendons, experience bony impingement due to normal anatomical positioning1,2,3,4. Tendon impingement generates compressive strain directed transversely to the longitudinal fiber axis5,6,7. Regions of tendon impingement demonstrate a unique fibrocartilage phenotype in which shrunken, round cells (fibrochondrocytes) are embedded within a disorganized collagen network with markedly increased glycosaminoglycan (GAG) content2,3,4,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24. Prior studies suggest the disparate mechanical environment produced by tendon impingement sustains this GAG-rich matrix by driving the deposition of large aggregating proteoglycans, most notably aggrecan, although the underlying mechanisms are unclear1,3,12,13,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39. While fibrocartilage is a normal feature in impinged regions of healthy tendons, aberrant proteoglycan metabolism associated with excessive fibrocartilage formation is a hallmark feature of tendinopathy, a common and debilitating disease that disproportionately emerges in chronically impinged tendons1,40,41,42,43,44,45,46,47,48,49. Accordingly, tendon impingement is clinically recognized as an important extrinsic factor driving several of the most common tendinopathies, including rotator cuff disease and insertional Achilles tendinopathy (IAT)50,51,52. Currently, treatment of tendinopathy is inefficient. For example, approximately 47% of patients with IAT require surgical intervention after failed conservative management, with variable postoperative outcomes53,54,55,56. Despite the apparent relationship between impingement and tendinopathy, the mechanobiological mechanisms by which cells in impinged tendon sense and respond to their mechanical environment are poorly described, which obscures understanding of tendinopathy pathogenesis and results in inadequate treatment.
Explant models are useful tools in the study of tendon mechanobiology57,58. As a first step towards understanding the mechanobiology of tendon impingement, several prior studies have explored cellular response following the application of simple uniaxial compression to cells or excised tendon explants27,29,30,31,32,33,34,39. However, cells in vitro lack extracellular and pericellular matrices that facilitate strain transfer, sequester important growth factors and cytokines released by mechanical deformation, and provide substrate for focal adhesion complexes that play a role in mechanotransduction57,59. Additionally, both in vitro and excised explant studies fail to recapitulate the multiaxial mechanical strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region5,6. In the context of the impinged Achilles tendon insertion, this includes surrounding tissues such as the retrocalcaneal bursa and Kager&#39;s fat pad60,61,62,63. Conversely, in vivo models of tendon impingement25,28,36,37,38,64,65,66 allow minimal control over the magnitude and frequency of load applied directly to the tendon, which is a well-recognized limitation of in vivo models for studying tendon mechanobiology57,58,67,68,69,70. Given challenges in measuring tendon strain in vivo, the internal strain environment generated within these models is often poorly characterized.
In this manuscript, we present a custom experimental platform that recreates impingement of the Achilles tendon insertion upon the calcaneus within whole murine hind limb explants that, when paired with this tissue culture protocol, maintains viability over 7 days in explant culture and allows for study of the biologic sequelae of tendon impingement. The platform is built upon a 3D printed polylactic acid (PLA) base that provides the foundation for the attachment of the grips and 3D printed PLA volume reduction insert. The grips are used to clamp the upper leg and knee proximal to the Achilles myotendinous junction with the caudal aspect of the hind limb facing upward, allowing the Achilles tendon to be imaged from above using an ultrasound probe or inverted microscope (Figure 1A). The volume reduction insert&#160;slides along a track on the base and reduces the required volume of tissue culture media. A braided line wrapped around the hind paw is routed out of the platform utilizing the base design and a 3D printed PLA clip. By pulling on the string, the hind paw is dorsiflexed, and the Achilles tendon insertion is impinged against the calcaneus, resulting in elevated transverse compressive strain5,6 (Figure 1A). The platform is contained within an acrylic bath that maintains the hind limb explants submerged in tissue culture media. Securing the taut string to the outside of the bath with adhesive tape maintains ankle dorsiflexion to produce static impingement of the Achilles tendon insertion. CAD files for 3D printed components are provided in multiple formats (Supplementary File 1), allowing import into a range of commercial and free, open-source CAD software for modification to suit experimental needs. If access to 3D printers is not available for fabrication, CAD files can be provided to online 3D printing services that will print and ship the parts at low cost.
Importantly, the triceps surae-Achilles musculotendinous complex spans both the knee and ankle joints71,72,73. Consequently, tensile strain in the Achilles tendon is influenced by knee flexion. Knee extension places the Achilles tendon under tension, whereas knee flexion reduces tension. By first extending the knee and then passively dorsiflexing the ankle, compressive strains at the impinged insertion can be superimposed upon tensile strains. Conversely, by passively dorsiflexing the ankle with the knee flexed, tensile strain is reduced, and compressive strain remains. The current protocol explores three such conditions. 1) For static impingement, the foot is dorsiflexed to &#60; 110&#176; with respect to the tibia to impinge the insertion, with the knee flexed to reduce tension. 2) For the baseline tension group, the ankle is extended above 145&#176; of dorsiflexion with the knee extended, generating predominately tensile strain at the insertion. 3) For the unloaded group, explants are cultured in a Petri dish with the knee and ankle in neutral positions in the absence of externally applied load. The angles referred to above are photographically measured relative to a coordinate system where the foot and tibia are parallel at an angle of 180&#176; and perpendicular at an angle of 90&#176;.
Key steps of the protocol include 1) dissection of hind limb explants and careful removal of the skin and plantaris tendon; 2) explant culture following a 48 h dexamethasone pretreatment; 3) tissue sectioning and histological staining; and 4) color image analysis to assess fibrocartilage formation. Following dissection, each hind limb explant is pretreated for 48 h in culture media&#160;supplemented with dexamethasone74. Contralateral limbs from each mouse are assigned to separate experimental groups for pairwise comparison, which helps control biological variability. After pretreatment, explants are positioned into platforms as described above and cultured for 7 more days (Figure 1B). Additional comparisons are made to a pretreated (day 0) group in which explants are removed immediately following the 48 h pretreatment.
After explant culture, hind limbs are trimmed down, formalin fixed, decalcified and embedded in paraffin. Serial sectioning in sagittal orientation provides visualization of the Achilles tendon from the myotendinous junction to the calcaneal insertion while allowing section depth to be tracked through the entire tendon. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP X-nick labeling (TUNEL) is used to visualize DNA damage secondary to apoptosis and assess viability. Toluidine blue histology and custom color image analysis are performed to quantify changes in GAG staining. Toluidine blue stained tissue sections are then used for SHG imaging to characterize alterations in collage fiber organization (Figure 1B).
The provided representative results suggest altered histological staining of the GAG-rich matrix and disorganization of the extracellular collagen network generated by 7 days of static impingement within the model. This model can be utilized to explore molecular mechanisms underlying impingement-driven fibrocartilaginous change.

作者聚焦：揭开肌腱撞击的力学生物学 – 多轴鼠后肢外植体模型 

小鼠后肢外植体研究跟腱撞击力学生物学的模型

We study the mechanobiology underlying tendon impingement and the process by which this unique mechanical demand drives localized fiber cartilage formation in health and disease. Here we seek to characterize matrix for modeling relative to spatially heterogeneous patterns of multiaxial mechanical strain, generated by impingement and to identify molecular mechanisms mediating this response. In vitro models for studying impingement mechanobiology have applied simple compression to isolated tendon cells or artificial uniaxial compression to partial and whole tendon explants.Established animal models of tendon impingement, manipulating external source of tendon impingement in vivo most often surgically and explore biology following resuming physical activity. In vitro models present significant limitations as isolated cells lack their three dimensional extracellular environment, crucial to mechano-response. While excised explant models circumvent this limitation, both fail to recreate multiaxial strain patterns generated by impingement in vivo.Conversely, animal models offer limited ability to measure or control internal tissue strains. Our Murine Hind Limb Explant Model for studying impingement mechanobiology maintains cells within their extracellular environment and preserves local anatomy of the impinged achilles tendon insertion in situ, allowing controlled prescription of impingement through passively applied joint motion to recreate multiaxial patterns of tissue strain that are measurable and well-characterized.

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?...

Watch this Scientific Journal Video about Unraveling the Mechanobiology of Tendon Impingement – A Multiaxial Murine Hind Limb Explant Model at JoVE.com

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。

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

我们研究了肌腱撞击的机械生物学，以及这种独特的机械需求在健康和疾病中驱动局部纤维软骨形成的过程。在这里，我们试图表征矩阵，以模拟相对于撞击产生的多轴机械应变的空间异质模式，并确定介导这种响应的分子机制。用于研究撞击机械生物学的体外模型已对分离的肌腱细胞进行简单压缩，或对部分和整个肌腱外植体进行人工单轴压缩。建立了肌腱撞击的动物模型，在体内操纵肌腱撞击的外部来源，最常通过手术，并在恢复身体活动后探索生物学。体外模型存在显着的局限性，因为分离的细胞缺乏对机械反应至关重要的三维细胞外环境。虽然切除的外植体模型规避了这一限制，但两者都无法在体内重现由撞击产生的多轴应变模式。相反，动物模型测量或控制内部组织菌株的能力有限。我们用于研究撞击机制生物学的小鼠后肢外植体模型将细胞维持在其细胞外环境中，并在原位保留撞击跟腱插入的局部解剖结构，允许通过被动应用的关节运动来控制撞击处方，以重建可测量且表征良好的组织应变的多轴模式。

murine-hind-limb-explant-model-for-studying-mechanobiology-achilles

Research

JoVE Journal

Bioengineering

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

科研

生物工程

Explant Dissection to Generate Murine Hind Limb Explant Model to Study Tendon Impingement

To begin, transfer 70 milliliters of pre-warmed Dulbecco's Modified Eagle Medium into a Petri dish in a sterile biosafety cabinet and add dexamethasone at a concentration of 100 nano molar. Assemble the surgical tools required for the dissection, which includes smooth, straight, fine tip forceps. Straight fine tip forceps with serrated teeth and straight sharp fine scissors.To dissect hind limb explants. Locate the hip joint of a euthanized mouse positioned supine on the bench top lined with absorbent under pads. Use fine scissors to create a small incision on the skin covering the proximal and anterior aspects of the upper leg.Then use fingers to pull the incision apart to expand and pinch the exposed upper leg. Carefully pull the skin distally to de-glove the hind limb to the level of the ankle. Insert one scissor blade gently under the skin along the dorsal aspect of the foot.Then make an incision that extends towards the toes and continue pulling the skin distally until it is completely removed. Position the mouse to have a clear view of the Achilles tendon insertion onto the posterior aspect of the calcaneus near the ankle. To remove the plant terrace tendon, insert one tip of the smooth fine tip forceps between the two tendons.Pass the tip medially under the plantaris tendon. Then pull it proximally to tear through the plantaris muscle. Then using fine tip serrated forceps, grasp the detached proximal end of the plantaris tendon and pull it distally to completely remove it.At the hip joint, employ fine scissors to precisely sever through the pelvis, isolating the hind limb. Use the scissors to gently remove the remaining pelvis, revealing the femoral head. In the biosafety cabinet transfer the hind limb explant into the dish containing cell culture media and dexamethasone.Relocate the dish to the incubator and subject it to pre-treatment for 48 hours.

外植体解剖生成小鼠后肢外植体模型以研究肌腱撞击

Explant Culture and Platforms Loading to Study the Biologic Sequelae of Tendon Impingement

To begin, preheat an adequate amount of sterile culture media while the 48-hour dexamethasone pretreatment of murine hindlimb explant is about to complete. Once the pretreatment ends, move the pretreated explants to the biosafety cabinet. For the unloaded group, aspirate the pretreatment media and transfer the explants to fresh Petri dishes.Add 70 milliliters of culture media to each dish before returning them to the incubator. To prepare the explant platforms for the baseline tension and static impingement group, cut pieces of sandpaper to match the size of the grip platens. For the static impingement group, cut pieces of braided line and pret tie a loose overhand knot halfway along the length of the line.Tear pieces of aluminum foil to cover each acrylic bath and spray them with 70%ethanol. Transfer the prepared components to the biosafety cabinet. Arrange the components such as acrylic bath, base, volume reduction inserts, clip, and grips for platform assembly.Place platform components inside secondary containers of sufficient volume to contain any potential culture media leaks. Submerge the containers in a solution of at least 10%bleach for a minimum of one hour. Rinse the bleach solutions with tap water.After transferring the rinsed components to the biosafety cabinet, use M3 screws and compression springs to affix the platens to the grips. Securely attach the grips to the base employing an M5 screw and insert the M6 screws until they contact the platens. Enhance the platens'functionality by affixing sandpaper using double-sided tape.Close the grips to facilitate the adhesion of the sandpaper to the platens. To load a platform, fully open the grips and use forceps to carefully position the upper leg and knee between the platens. Close the grips gently to hold the limb in place.Use forceps to grasp the exposed femoral head or foot and adjust the knee flexion angle, while gradually closing the forceps to secure it in place. For the baseline tension group, extend the knee joint while closing the grips. In the case of the static impingement group, flex the knee joint while closing the grips.For the static impingement group, place the overhand knot of the string around the distal paw and tighten it securely. Route the string through a slot in the base underneath the explant and through the clip hole. Position the base into the acrylic bath and fasten the clip to the top edge of the bath.To dorsiflex the foot, pull the string until it reaches at least 110 degrees relative to the tibia. Use a permanent marker to mark the string as it exits the clip. Capture a photograph of the explant in this dorsiflexed position for future quantification of the dorsiflexion angle.Remove the base from the setup and attach the volume reduction insert by sliding it along a track on the base. Place the base back into the acrylic bath and reposition the clip on the top edge. Use the marked string as a guide.Pull it to restore the foot to its original dorsiflexion angle and secure the string to the exterior of the bath using tape to maintain a static dorsiflexed position. For the baseline tension group, once the explant is positioned between the grips, take a photograph to quantify the dorsiflexion angle. Then slide the volume reduction insert onto the base and place it into the acrylic bath.Add 125 milliliters of prewarmed culture media to each platform to submerge the explants. After covering the top of the bath with aluminum foil, place the entire setup into a secondary container. Relocate the secondary container to the incubator and culture for seven days.