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

Compreendendo o impacto do tendão - Modelo de explante murino para estudar mecanobiologia

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 em destaque: Desvendando a mecanobiologia do impacto do tendão - um modelo multiaxial de explante de membros posteriores murinos 

Modelo de Explante de Membro Posterior Murino para Estudo da Mecanobiologia do Impacto do Tendão de Aquiles

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  

Cultura de Explantes e Carregamento de Plataformas para Estudo das Sequelas Biológicas do Impacto Tendíneo

Para começar, pré-aqueça uma quantidade adequada de meios de cultura estéreis enquanto o pré-tratamento de 48 horas com dexametasona do explante murino dos membros posteriores está prestes a ser concluído. Terminado o pré-tratamento, mova os explantes pré-tratados para o gabinete de biossegurança. Para o grupo descarregado, aspirar o meio de pré-tratamento e transferir os explantes para placas de Petri frescas.Adicione 70 mililitros de meios de cultura a cada prato antes de devolvê-los à incubadora. Para preparar as plataformas de explante para o grupo de tensão basal e impacto estático, corte pedaços de lixa para corresponder ao tamanho das placas de aderência. Para o grupo de impacto estático, corte pedaços de linha trançada e amarre um nó overhand solto no meio do comprimento da linha.Rasgue pedaços de papel alumínio para cobrir cada banho de acrílico e borrife com etanol 70%. Transfira os componentes preparados para o gabinete de biossegurança. Organize os componentes como banho de acrílico, base, insertos de redução de volume, grampo e pegas para montagem da plataforma.Coloque os componentes da plataforma dentro de recipientes secundários de volume suficiente para conter possíveis vazamentos de meios de cultura. Submergir os recipientes em uma solução de pelo menos 10% de água sanitária por um período mínimo de uma hora. Enxágue as soluções de água sanitária com água da torneira.Após transferir os componentes enxaguados para o gabinete de biossegurança, utilize parafusos M3 e molas de compressão para fixar as placas nas alças. Fixe firmemente as alças à base empregando um parafuso M5 e insira os parafusos M6 até que entrem em contato com as placas. Melhore a funcionalidade das placas afixando lixas usando fita dupla face.Feche as alças para facilitar a aderência da lixa às placas. Para carregar uma plataforma, abra totalmente as alças e use pinças para posicionar cuidadosamente a parte superior da perna e o joelho entre as placas. Feche as alças suavemente para manter o membro no lugar.Use a pinça para agarrar a cabeça ou o pé femoral exposto e ajuste o ângulo de flexão do joelho, fechando gradualmente a pinça para prendê-la no lugar. Para o grupo de tensão basal, estenda a articulação do joelho enquanto fecha as pegas. No caso do grupo de impacto estático, flexione a articulação do joelho enquanto fecha as pegas.Para o grupo de impacto estático, coloque o nó overhand da corda ao redor da pata distal e aperte-a com segurança. Direcione a corda através de um slot na base abaixo do explante e através do orifício do clipe. Posicione a base no banho de acrílico e prenda o clipe na borda superior do banho.Para dorsiflexar o pé, puxe a corda até atingir pelo menos 110 graus em relação à tíbia. Use um marcador permanente para marcar a cadeia de caracteres ao sair do clipe. Capturar uma fotografia do explante nesta posição dorsiflexionada para futura quantificação do ângulo de dorsiflexão.Remova a base da configuração e conecte a pastilha de redução de volume deslizando-a ao longo de uma esteira na base. Coloque a base de volta no banho de acrílico e reposicione o clipe na borda superior. Use a cadeia de caracteres marcada como guia.Puxe-o para restaurar o pé ao seu ângulo de dorsiflexão original e prenda a corda ao exterior do banho usando fita adesiva para manter uma posição dorsiflexada estática. Para o grupo de tensão basal, uma vez posicionado o explante entre as pegas, fotografe para quantificar o ângulo de dorsiflexão. Em seguida, deslize a pastilha de redução de volume sobre a base e coloque-a no banho de acrílico.Adicione 125 mililitros de meios de cultura pré-aquecidos a cada plataforma para submergir os explantes. Depois de cobrir a parte superior do banho com papel alumínio, coloque todo o setup em um recipiente secundário. Realocar o recipiente secundário para a incubadora e cultura por sete dias.

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 ...