Name: Explant Culture and Platforms Loading to Study the Biologic Sequelae of Tendon Impingement
Uploaded: 2023-12-08T13:30:00
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explant culture and platforms loading to study the biologic sequelae of tendon impingement

了解肌腱撞击 – 用于研究机械生物学的小鼠外植体模型

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.

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

外植体培养和平台加载以研究肌腱撞击的生物学后遗症

首先，预热足量的无菌培养基，同时鼠后肢外植体的48小时地塞米松预处理即将完成。预处理结束后，将预处理的外植体移至生物安全柜中。对于卸载组，吸出预处理培养基并将外植体转移到新鲜培养皿中。在将培养基放回培养箱之前，向每个培养皿中加入 70 毫升培养基。为了准备基线张力和静态冲击组的外植体平台，切割砂纸片以匹配夹板的尺寸。对于静态冲击组，剪下编织线的碎片，并沿着线的长度在中间系一个松散的反手结。撕下铝箔片覆盖每个丙烯酸浴槽，并喷洒70%乙醇。将准备好的组件转移到生物安全柜中。布置组件，例如亚克力浴缸、底座、体积减小插件、夹子和用于平台组装的把手。将平台组件放入足够体积的二级容器中，以容纳任何潜在的培养基泄漏。将容器浸入至少 10% 漂白剂的溶液中至少一小时。用自来水冲洗漂白剂溶液。将冲洗后的组件转移到生物安全柜后，使用 M3 螺钉和压缩弹簧将压板固定到把手上。使用 M5 螺钉将夹具牢固地固定到底座上，然后插入 M6 螺钉，直到它们接触压板。通过使用双面胶带粘贴砂纸来增强压板的功能。关闭把手以促进砂纸与压板的粘附。要装载平台，请完全打开把手并使用镊子小心地将大腿和膝盖放在压板之间。轻轻合上把手以将肢体固定到位。用镊子抓住暴露的股骨头或脚，调整膝关节屈曲角度，同时逐渐闭合镊子以将其固定到位。对于基线张力组，在闭合握把的同时伸展膝关节。在静态冲击组的情况下，在关闭握把的同时弯曲膝关节。对于静态冲击组，将绳子的反手结放在远端爪子周围并牢固地拧紧。将绳子穿过外植体下方底座上的槽并穿过夹孔。将底座放入丙烯酸浴中，并将夹子固定在浴槽的顶部边缘。要背屈脚，请拉动绳子，直到它相对于胫骨至少达到 110 度。当绳子离开夹子时，使用永久性记号笔标记绳子。在此背屈位置拍摄外植体的照片，以便将来量化背屈角。从设置中取出底座，然后沿着底座上的轨道滑动来安装体积减小插件。将底座放回丙烯酸浴中，然后将夹子重新定位在顶部边缘。使用标记的字符串作为指导。拉动它以将脚恢复到原来的背屈角度，并使用胶带将绳子固定在浴缸的外部以保持静态的背屈位置。对于基线张力组，一旦外植体位于握把之间，请拍照以量化背屈角。然后将体积减小插件滑到底座上，并将其放入丙烯酸浴中。向每个平台中加入 125 毫升预热的培养基以浸没外植体。用铝箔覆盖浴缸顶部后，将整个装置放入辅助容器中。将辅助容器移至培养箱并培养 7 天。

Research

JoVE Journal

Bioengineering

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

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 ...