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

Förstå senimpingement – Murine Explantation-modell för att studera mekanobiologi

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.

Författare Spotlight: Att reda ut mekanobiologin för senimpingement - En multiaxial murin bakbens explantationsmodell 

Murin bakbensexplantationsmodell för att studera mekanobiologin vid impingement av akillessenan

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  

Explantkultur och plattformsladdning för att studera de biologiska följderna av senpåverkan

Till att börja med ska du förvärma en tillräcklig mängd sterilt odlingsmedium medan 48-timmars förbehandlingen av murina bakbensexplantor är på väg att slutföras. När förbehandlingen är klar flyttar du den förbehandlade explanten till biosäkerhetsskåpet. För den avlastade gruppen, aspirera förbehandlingsmediet och överför explanten till färska petriskålar.Tillsätt 70 ml odlingsmedia till varje maträtt innan du lägger tillbaka dem i inkubatorn. För att förbereda explanteringsplattformarna för baslinjespänningen och den statiska impingementgruppen, skär bitar av sandpapper så att de matchar storleken på greppplattorna. För den statiska impingementgruppen, klipp bitar av flätad lina och knyt en lös överhandsknut halvvägs längs linans längd.Riv bitar av aluminiumfolie för att täcka varje akrylbad och spraya dem med 70% etanol. Överför de förberedda komponenterna till biosäkerhetsskåpet. Ordna komponenter som akrylbad, bas, volymreducerande insatser, klämmor och grepp för plattformsmontering.Placera plattformskomponenter i sekundära behållare med tillräcklig volym för att innehålla eventuella läckage av odlingsmedier. Sänk ner behållarna i en lösning av minst 10 % blekmedel i minst en timme. Skölj blekmedelslösningarna med kranvatten.Efter att ha överfört de sköljda komponenterna till biosäkerhetsskåpet, använd M3-skruvar och tryckfjädrar för att fästa plattorna på handtagen. Fäst handtagen ordentligt på basen med en M5-skruv och sätt i M6-skruvarna tills de kommer i kontakt med plattorna. Förbättra plattornas funktionalitet genom att fästa sandpapper med dubbelhäftande tejp.Stäng handtagen för att underlätta sandpapprets vidhäftning till plattorna. För att ladda en plattform, öppna handtagen helt och använd pincett för att försiktigt placera överbenet och knäet mellan plattorna. Stäng handtagen försiktigt för att hålla lemmen på plats.Använd en pincett för att ta tag i det exponerade lårbenshuvudet eller lårbensfoten och justera knäböjningsvinkeln, samtidigt som du gradvis stänger pincetten för att säkra den på plats. För baslinjespänningsgruppen, sträck ut knäleden samtidigt som du stänger greppen. När det gäller den statiska impingementgruppen, böj knäleden samtidigt som du stänger handtagen.För den statiska impingementgruppen, placera snörets överhandsknut runt den distala tassen och dra åt den ordentligt. Dra snöret genom en skåra i basen under explanten och genom klämhålet. Placera basen i akrylbadet och fäst klämman i badkarets överkant.För att dorsalflexa foten, dra i snöret tills det når minst 110 grader i förhållande till skenbenet. Använd en permanent markör för att markera strängen när den lämnar klippet. Ta ett fotografi av explanten i denna dorsalflexerade position för framtida kvantifiering av dorsalflexionsvinkeln.Ta bort basen från installationen och fäst volymreduceringsinsatsen genom att skjuta den längs ett spår på basen. Sätt tillbaka basen i akrylbadet och sätt tillbaka klämman på den övre kanten. Använd den markerade strängen som vägledning.Dra i den för att återställa foten till sin ursprungliga dorsalflexionsvinkel och fäst snöret på utsidan av badet med tejp för att bibehålla en statisk dorsalflexerad position. För baslinjespänningsgruppen, när explanten är placerad mellan greppen, ta ett fotografi för att kvantifiera dorsalflexionsvinkeln. Skjut sedan volymreduceringsinsatsen på basen och placera den i akrylbadet.Tillsätt 125 milliliter förvärmda odlingsmedier till varje plattform för att sänka ner explanterna. Efter att ha täckt toppen av badet med aluminiumfolie, placera hela installationen i en sekundär behållare. Flytta den sekundära behållaren till inkubatorn och kulturen i sju dagar.

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 ...