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

Verständnis des Sehnenimpingements – Mausexplantatmodell zur Untersuchung der Mechanobiologie

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.

Autor im Rampenlicht: Entschlüsselung der Mechanobiologie des Sehnenimpingements – Ein multiaxiales Explantatmodell für die Hintergliedmaßen der Maus 

Maus-Hintergliedmaßen-Explantatmodell zur Untersuchung der Mechanobiologie des Achillessehnen-Impingements

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.

Watch this Scientific Journal Video about Explant Culture and Platforms Loading to Study the Biologic Sequelae of Tendon Impingement at JoVE.com

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

Explantatskultur und Plattformbelastung zur Untersuchung der biologischen Folgen des Sehnenimpingements

Zu Beginn wird eine ausreichende Menge steriler Nährmedien vorgewärmt, während die 48-stündige Dexamethason-Vorbehandlung der männlichen Hintergliedmaßenexplantation kurz vor dem Abschluss steht. Sobald die Vorbehandlung abgeschlossen ist, bringen Sie die vorbehandelten Explantate in die Biosicherheitswerkbank. Für die entladene Gruppe wird das Vorbehandlungsmedium abgesaugt und die Explantate in frische Petrischalen überführt.Geben Sie 70 Milliliter Nährmedien in jede Schale, bevor Sie sie in den Inkubator zurückgeben. Um die Explantatsplattformen für die Grundspannungs- und statische Aufprallgruppe vorzubereiten, schneiden Sie Schleifpapierstücke auf die Größe der Griffplatten. Für die statische Impingement-Gruppe schneiden Sie Stücke der geflochtenen Schnur ab und binden einen lockeren Überhandknoten auf halber Höhe der Schnur.Reißen Sie Stücke der Aluminiumfolie ab, um jedes Acrylbad abzudecken, und besprühen Sie sie mit 70%igem Ethanol. Übergeben Sie die vorbereiteten Komponenten in die Biosicherheitswerkbank. Ordnen Sie die Komponenten wie Acrylbad, Sockel, Volumenreduzierungseinsätze, Clip und Griffe für die Plattformmontage an.Platzieren Sie die Plattformkomponenten in sekundären Behältern mit ausreichendem Volumen, um mögliche Leckagen in den Nährmedien einzudämmen. Tauchen Sie die Behälter mindestens eine Stunde lang in eine Lösung von mindestens 10 % Bleichmittel. Spülen Sie die Bleichlösungen mit Leitungswasser ab.Nachdem Sie die gespülten Komponenten in die Biosicherheitswerkbank überführt haben, verwenden Sie M3-Schrauben und Druckfedern, um die Platten an den Griffen zu befestigen. Befestigen Sie die Griffe mit einer M5-Schraube sicher an der Basis und setzen Sie die M6-Schrauben ein, bis sie die Platten berühren. Verbessern Sie die Funktionalität der Platten, indem Sie Schleifpapier mit doppelseitigem Klebeband anbringen.Schließen Sie die Griffe, um das Anhaften des Schleifpapiers auf den Platten zu erleichtern. Um eine Plattform zu laden, öffnen Sie die Griffe vollständig und positionieren Sie mit einer Pinzette vorsichtig den Oberschenkel und das Knie zwischen den Platten. Schließen Sie die Griffe vorsichtig, um die Gliedmaße an Ort und Stelle zu halten.Verwenden Sie eine Pinzette, um den freiliegenden Hüftkopf oder -fuß zu fassen und den Kniebeugewinkel einzustellen, während Sie die Pinzette allmählich schließen, um sie an Ort und Stelle zu halten. Für die Grundlinien-Spannungsgruppe streckst du das Kniegelenk aus, während du die Griffe schließt. Bei der statischen Impingementgruppe beugen Sie das Kniegelenk beim Schließen der Griffe.Für die statische Impingementgruppe legen Sie den Überhandknoten der Schnur um die distale Pfote und ziehen ihn fest an. Führen Sie die Schnur durch einen Schlitz in der Basis unter dem Explantat und durch das Cliploch. Positionieren Sie den Sockel in der Acrylwanne und befestigen Sie den Clip an der Oberkante der Badewanne.Um den Fuß zu beugen, ziehen Sie an der Sehne, bis sie mindestens 110 Grad relativ zum Schienbein erreicht. Verwende einen Permanentmarker, um die Zeichenfolge beim Verlassen des Clips zu markieren. Nehmen Sie ein Foto des Explantats in dieser dorsalflexierten Position auf, um den Dorsalflexionswinkel später zu quantifizieren.Nehmen Sie den Sockel aus dem Setup und befestigen Sie den Volumenreduzierungseinsatz, indem Sie ihn entlang einer Schiene auf dem Sockel schieben. Setzen Sie den Sockel wieder in das Acrylbad ein und positionieren Sie den Clip an der oberen Kante neu. Verwenden Sie die markierte Zeichenfolge als Anhaltspunkt.Ziehen Sie daran, um den Fuß wieder in seinen ursprünglichen Dorsalflexionswinkel zu bringen, und befestigen Sie die Schnur mit Klebeband an der Außenseite der Badewanne, um eine statische dorsalflexierte Position beizubehalten. Sobald das Explantat zwischen den Griffen positioniert ist, machen Sie für die Basislinienspannungsgruppe ein Foto, um den Dorsalflexionswinkel zu quantifizieren. Schieben Sie dann den Volumenreduzierungseinsatz auf den Sockel und legen Sie ihn in das Acrylbad.Geben Sie 125 Milliliter vorgewärmtes Nährmedium zu jeder Plattform, um die Explantate einzutauchen. Nachdem Sie die Oberseite des Bades mit Aluminiumfolie abgedeckt haben, geben Sie das gesamte Setup in einen sekundären Behälter. Bringen Sie den sekundären Behälter für sieben Tage in den Inkubator und die Kultur.

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 ...