Intrinsic and Extrinsic Healing Mechanisms in Tendon: Crosstalk and Regenerative Potential

Tendons are the organs transferring muscle forces to the bones to enable locomotion. Consequently, populations suffering from tendon-related diseases often share a history of repetitive mechanical overloading and include heavy manual laborers, professional athletes, the obese, and the elderly. The prevalence of tendon-related diseases is projected to rise in Western countries due to their aging societies, increasing obesity, and the popularity of mechanically challenging sport activities. Tendinopathy is the most common tendon-related disease. So far, multiple roadblocks have largely prevented the development of evidence-based and specifically disease-modifying treatment and relapse-preventing regimes for tendinopathy. Since early tendinopathy is often asymptomatic, in vivo human studies only compare supposedly healthy to end-stage diseased tendons and therefore fail to longitudinally capture the pathogenic mechanisms. In in vivo murine models, it is challenging to dissect specific bi- or multicellular interaction pathways triggered by a defined microenvironmental stress (disease, damage, age). While this in vivo complexity prevents fast treatment screening and evaluation, simple traditional 2D model systems generally fail to adequately recapitulate the central tendon function – multi-dimensional mechanical loading including tension (in the direction of the tendon loading), compression (vertically to the direction of tendon loading), and shear from fiber sliding. Tissue-engineered 3D ex vivo model systems could fill this gap, but often lack loadability over extended periods of time and fall short of replicating the in vivo extracellular matrix informing cell behavior. Full murine tendons explants (e.g. Achilles or patellar tendon) on the other hand are small, which hampers clamping reproducibility and collection of sufficient material for cell-, protein-, and gene-level readouts. Murine tail tendon fascicle explants are readily available in large numbers, recapitulate the complex in vivo loading patterns, and possess an in vivo-like extracellular matrix composition but largely lack vascular, immune, and progenitor cell populations present in the so-called extrinsic tendon compartment. The aim of this dissertation was to increase the applicability of murine tail tendon fascicles by fabricating a high-throughput drug testing system mimicking the initial stages of the tendinopathic cascade (overloading, microdamage, inflammation). Conceptually, we divided the tendons repair response into three overlapping parts: the mechanisms governing the intrinsic, tendon core-mediated tendon maintenance and response to microdamage, the danger-signaling pathways deployed by an overwhelmed tendon core to recruit the extrinsic compartment, and ultimately the lesion-healing response mounted by components of the recruited extrinsic compartment. First, we studied the effect of mechanically induced microdamage on tendon core explants in conditions mimicking a quiescent, healthy tendon niche. We created and characterized different levels of functional, structural, and cellular microdamage, which revealed the core’s limited regenerative capabilities. Second, we sequentially added populations of the extrinsic compartment to the model system to characterize their effects on the tendon core explant and emerging cross-compartmental communication. Since the cross-compartmental handshaking has been hypothesized to occur post-damage, these experiments were performed in conditions mimicking an injury-like tendon niche. The resulting hybrid explant // hydrogel assembloid model (which we termed “tenostruct”) recapitulates key post-injury events like degeneration of underloaded core tissue, emigration of a newly discovered core-resident tenoclast population, extrinsic tendon-lineage cells recruiting to the degenerating core to accelerate its degeneration, and extrinsic macrophages differentiating to mitigate the degeneration. Third, we looked further into the cytokine signatures in degrading versus non-degrading tenostructs and identified interleukin-6 (IL-6) as a major differentiating factor. Leveraging the modularity of the tenostructs, we incorporated core tissue from a mouse strain unable to produce interleukin-6 (so-called IL-6 knock-out mice) or added an IL-6 inhibitor to the culture media and found reductions in tendon lineage progenitor recruitment, overall cell proliferation, and degeneration of an IL-6 knock-out core. Finally, we confirmed the upregulation of IL-6 signaling pathways in diseased compared to healthy human samples with microarray data. In summary, we establish a novel hybrid hydrogel // explant assembloid model which allows the user to study the effects of mechanical, metabolic, and matrix-based stressors on construct behaviors ranging from compartment-specific gene expression to mechanical properties. The model easily integrates tissues and cells from genetically modified animals to follow the contribution of a specific pathway in disease progression. We demonstrate the power of the model system by combining it with human data and dissecting the role of IL-6 signaling in tendon lesion progression.Die Hauptfunktion des Sehnenorgans ist die Übertragung von Muskelkräften auf die Knochen um so Bewegungen zu ermöglichen. Aus diesem Grund teilen Patienten mit Sehnenläsionen oft eine Vergangenheit mit wiederholten mechanischen Sehnenüberbelastungen. Untergruppen dieser Patienten umfassen Berufsgattungen die schwere körperliche Arbeit verrichten, professionelle Athleten, übergewichtige Menschen und ältere Menschen. Westlichen Gesellschaften wird aufgrund ihrer Überalterung, dem erhöhten Anteil von übergewichtigen oder gar fettleibigen Individuen und einer immer weiter verbreiterten Popularität von mechanisch herausfordernden Sportarten eine zunehmende Prävalenz von Sehnenläsionen vorausgesagt. Tendinopathien stellen die geläufigste Art von Sehnenläsionen dar. Leider wurde die Entwicklung von evidenzbasierten, krankheitsspezifischen und -modulierenden Behandlungen und rückfallverhindernden Therapien für Tendinopathien bis jetzt durch zahlreiche Barrieren verlangsamt. Da sich Tendinopathien beispielsweise im Frühstadium oft symptomlos präsentieren, können in Menschen durchgeführte Studien nur Gewebe von angeblich gesunden Sehnen mit solchem von weit fortgeschrittenen Krankheitsbildern vergleichen. Diese limitierte zeitliche Auflösung des Krankheitsverlaufs macht es fast unmöglich die Krankheitsentwicklung exakt nachzuvollziehen geschweige denn rückgängig zu machen. In vivo Modelsysteme basierend auf lebenden Nagetieren (wie Mäusen oder Ratten) könnten zwar theoretisch die Nachverfolgung der Krankheitsentwicklung erlauben, deren Komplexität macht es aber schwierig die Auswirkungen von spezifischen Zell-Zell-Interaktionen und Signalwegen isoliert zu studieren. Ausserdem sind Effekte möglicher Auslöser von Tendinopathien in Menschen (z.B. Krankheiten, überbelastungsinduzierter Gewebeschaden und hohes Alter) nur bedingt in Nagetieren induzierbar. Simple, traditionelle in vitro Modellsysteme wären dafür zwar besser geeignet, können aber aufgrund ihrer Zweidimensionalität die im Rahmen der normalen Sehnenfunktion auftretenden multi-dimensionalen Belastungskomponenten wie Zug-, Druck- und Scherkräfte nur ungenügend simulieren. Dreidimensionale in vitro Modelle basierend auf Explantaten oder gezüchtetem Gewebe könnten diese Lücke füllen. Gezüchtete Gewebe weisen aber noch nicht die nötige Stabilität für Langzeitbelastungsstudien auf und reproduzieren die in vivo die Zellen umgebende Matrix höchstens ansatzweise. Explantate zum Beispiel der Achilles oder Patellarsehne erhalten diese Matrix, ihre kurze Länge erschwert jedoch die reproduzierbare Einspannung für mechanische Stimulationen und das Sammeln von genügend Material für umfangreiche Messungen auf den Zell-, Protein- und Genebenen. Die hier verwendeten, aus dem Schwanz von Nagetieren isolierten Sehnenfaszikelexplantate hingegen sind einfach in genügend grossen Mengen zugänglich, können die komplexen in vivo Belastungskomponenten nachvollziehen und erhalten die in vivo Zusammensetzung der zellumgebenden Matrix. Allerdings verlieren diese Sehnenfaszikel, die in ihrer Summe als Sehnenkern zusammengefasst werden, bei der Explantation auch den Zugang zu einigen für die Läsionsheilung benötigten extrinsischen Zellpopulation wie Vorläuferzellen und Zellen des Immunsystems und des vaskulären Systems. Das Ziel dieser Dissertation war es, die aus dem Schwanz von Nagetieren isolierten Sehnenfaszikel für die Fabrikation eines Modellsystems zu testen, dass die frühen Stadien der Tendinopathie (Überbelastung, Gewebeschaden, Entzündungsreaktion) nachvollziehen kann. Dieses Modellsystem sollte sich dabei die einzigartigen Vorteile der Sehnenfaszikel zunutze machen um dann mit grossem Durchsatz Behandlungsmethoden sichten und evaluieren zu können. Konzeptuell unterteilten wir die von der Läsionsschwere abhängigen Reparaturmechanismen der Sehne in drei überlappende Bereiche: Die Mechanismen des für Wartungsarbeiten und Mikroläsionen zuständigen Sehnenkerns, dessen Alarmmechanismen, die bei Überforderung durch grössere Läsionen die Zellpopulationen des extrinsischen Kompartiments rekrutieren, und schliesslich die Läsionsheilungsmechanismen der Komponenten des rekrutierten extrinsischen Kompartiments. In einem ersten Schritt studierten wir die Auswirkungen von überlastungsinduziertem Gewebeschaden auf den Sehnenkern in Kulturbedingungen, die jenen einer gesunden Sehne mit niedriger metabolischer Aktivität nachempfunden waren. Wir erzeugten verschiedene Stufen von Gewebeschaden und charakterisierten deren Effekte auf die funktionellen und strukturellen Eigenschaften der Explantate sowie auf die sich darin befindenden Zellpopulationen. Diese Experimente offenbarten die unerwartet stark limitierten Fähigkeiten des Sehnenkerns zur Selbstreparatur. In einem zweiten Schritt fügten wir dem Sehnenkern nacheinander verschiedene, in einem Hydrogel eingebettete Zellpopulationen aus dem extrinsischen Kompartiment hinzu und charakterisierten die entstehenden wechselseitigen, kompartiments-übergreifenden Interaktionen. Das dabei entstandene hybride Modellsystem nannten wir «Tenostrukt» und exponierten es lädierten Sehnen nachempfundenen Kulturbedingungen, in welchen kompartiments-übergreifende Interaktionen eine entscheidende Rolle spielen. Dabei konnten wir zeigen, dass Tenostrukte fähig sind Schlüsselmerkmale von chronischen Sehnenläsionen zu rekapitulieren. Dazu gehören etwa die fortschreitende Degeneration von unterbelastetem Sehnenkerngewebe, die Migration einer neu entdeckten, sonst im Sehnenkern beheimateten Tenoklastpopulation ins extrinsische Kompartiment, die Rekrutierung von extrinsischen Sehnenlinienvorläuferzellen zum und eine daraus resultierende Degenerationsbeschleunigung im geschädigten Sehnenkern sowie die Differenzierung von und Verlangsamung der Degenerationgeschwindigkeit durch extrinsische Makrophagen. Im dritten Teilprojekt fokussierten wir uns auf Unterschiede in der Produktion von Zytokinen, signalübertragenden Peptiden und Proteinen zwischen degenerierenden und nicht-degenerierenden Tenostrukten und identifizierten dabei unter anderem Interleukin-6 (IL-6) als entscheidenden Faktor. Wir nutzten dann die Modularität der Tenostrukte und ersetzten die Wildtyp Explantate mit solchen von einer Mauslinie, die Interleukin-6 nicht herstellen kann (sogenannte IL-6 knock-out Mäuse) oder ergänzten das Zellkulturmedium mit einem Interleukin-6 Inhibitor. Diese Hemmung der IL-6 Signalübertragung führte zu Reduktionen in der Rekrutierung von Vorläuferzellen, der allgemeinen Zellproliferation und der Degeneration der IL-6 knock-out Explantate. Zum Schluss bestätigten wir die Relevanz der Interleukin-6 Signalübertragung in Tendinopathien indem wir die Genexpression von gesunden und lädierten menschlichen Sehnen miteinander verglichen. Zusammenfassend haben wir ein neuartiges, hybrides Modellsystem bestehend aus einem Hydrogel und einem Explantat entwickelt. Dieses Modelsystem erlaubt es seinen Nutzern, die Effekte von mechanischen, metabolischen und matrix-basierten Stressoren auf das Verhalten des Konstrukts zu untersuchen. Dieses Verhalten kann auf mehreren Ebenen quantifiziert werden: Von der kompartiments-spezifischen Genexpression über die Ausschüttung von Botenstoffen bis hin zu den mechanischen Eigenschaften. Die Modularität des Modellsystems erlaubt eine einfache Integration von Explantaten oder Zellen von genmodifizierten Tieren um dann den Einfluss des modifizierten Faktors auf die kompartiments-übergreifenden Interaktionen zu untersuchen. Wir demonstrierten das dahingehende Potential des neuen Modellsystems anhand der Rolle von Interleukin-6 Signalübertragungen in der Entstehung von Schlüsselmerkmalen der Tendinopathien und bestätigten deren Relevanz mit Daten aus menschlichem Gewebe

Tendon tissue microdamage and the limits of intrinsic repair

The transmission of mechanical muscle force to bone for musculoskeletal stability and movement is one of the most important functions of tendon. The load-bearing tendon core is composed of highly aligned collagen-rich fascicles interspersed with stromal cells (tenocytes). Despite being built to bear very high mechanical stresses, supra-physiological/repetitive mechanical overloading leads to tendon microdamage in fascicles, and potentially to tendon disease and rupture. To date, it is unclear to what extent intrinsic healing mechanisms of the tendon core compartment can repair microdamage. In the present study, we investigated the healing capacity of the tendon core compartment in an ex vivo tissue explant model. To do so, we isolated rat tail tendon fascicles, damaged them by applying a single stretch to various degrees of sub-rupture damage and longitudinally assessed downstream functional and structural changes over a period of several days. Functional damage was assessed by changes in the elastic modulus of the material stress-strain curves, and biological viability of the resident tenocytes. Structural damage was quantified using a fluorescent collagen hybridizing peptide (CHP) to label mechanically disrupted collagen structures. While we observed functional mechanical damage for strains above 2% of the initial fascicle length, structural collagen damage was only detectable for 6% strain and beyond. Minimally loaded/damaged fascicles (2-4% strain) progressively lost elastic modulus over the course of tissue culture, despite their collagen structures remaining intact with high degree of maintained cell viability. In contrast, more severely overloaded fascicles (6-8% strain) with damage at the molecular/collagen level showed no further loss of the elastic modulus but markedly decreased cell viability. Surprisingly, in these heavily damaged fascicles the elastic modulus partially recovered, an effect also seen in further experiments on devitalized fascicles, implying the possibility of a non-cellular but matrix-driven mechanism of molecular repair. Overall, our findings indicate that the tendon core has very little capacity for self-repair of microdamage. We conclude that stromal tenocytes likely do not play a major role in anabolic repair of tendon matrix microdamage, but rather mediate catabolic matrix breakdown and communication with extrinsic cells that are able to effect tissue repair

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease-and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.ISSN:1940-087

Tyrosinase-crosslinked, tissue adhesive and biomimetic alginate sulfate hydrogels for cartilage repair

The native cartilage extracellular matrix (ECM) is enriched in sulfated glycosaminoglycans with important roles in the signaling and phenotype of resident chondrocytes. Recapitulating the key ECM components within engineered tissues through biomimicking strategies has potential to improve the regenerative capacity of encapsulated cells and lead to better clinical outcome. Here, we developed a double-modified, biomimetic and tissue adhesive hydrogel for cartilage engineering. We demonstrated sequential modification of alginate with first sulfate moieties to mimic the high glycosaminoglycan content of native cartilage and then tyramine moieties to allow in situ enzymatic crosslinking with tyrosinase under physiological conditions. Tyrosinase-crosslinked alginate sulfate tyramine (ASTA) hydrogels showed strong adhesion to native cartilage tissue with higher bond strength compared to alginate tyramine (AlgTA). Both ASTA and AlgTA hydrogels supported the viability of encapsulated bovine chondrocytes and induced a strong increase in the expression of chondrogenic genes such as collagen 2, aggrecan and Sox9. Aggrecan and Sox9 gene expression of chondrocytes in ASTA hydrogels were significantly higher than those in AlgTA. Chondrocytes in both ASTA and AlgTA hydrogels showed potent deposition of cartilage matrix components collagen 2 and aggrecan after 3 weeks of culture whereas a decreased collagen 1 deposition was observed in the sulfated hydrogels. ASTA and AlgTA hydrogels with encapsulated human chondrocytes showed in vivo stability as well as cartilage matrix deposition upon subcutaneous implantation into mice for 4 weeks. Our data is the first demonstration of a double-modified alginate with sulfation and tyramination that allows in situ enzymatic crosslinking, strong adhesion to native cartilage and chondrogenic re-differentiation. (© 2020 IOP Publishing Ltd.

Extrinsic Macrophages Protect While Tendon Progenitors Degrade: Insights from a Tissue Engineered Model of Tendon Compartmental Crosstalk

Tendons are among the most mechanically stressed tissues of the body, with a functional core of type-I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load-bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial-like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell-laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146+ tendon stem/progenitor cell and CD45+ F4/80+ bone-marrow derived macrophage activity within a tendon injury-like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone-marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80+ "tenophages" from the intrinsic tissue core. Keywords: crosstalk; ex vivo tissue; macrophages; progenitors; tendonsTendons are among the most mechanically stressed tissues of the body, with a functional core of type-I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load-bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial-like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell-laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146$^{+}$ tendon stem/progenitor cell and CD45$^{+}$ F4/80$^{+}$ bone-marrow derived macrophage activity within a tendon injury-like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone-marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80$^{+}$ "tenophages" from the intrinsic tissue core

Robot- and Laser-Assisted Bio-Sample Preparation: Development of an Integrated, Intuitive System

The preparation of small-sized biological samples is traditionally performed manually utilizing mechanical tools such as scalpels. The main drawbacks of such methods are a lack of accuracy and repeatability of the resulting cuts and damage to the surrounding tissue due to the high interaction forces and the accompanying mechanical stresses. One way to circumvent these issues is to substitute the mechanical tools for laser light. When used in conjunction with a high-accuracy positioning system, such a preparation procedure enables repeatable cutting of arbitrary geometries while largely preserving the integrity of the surrounding tissue. In this paper, a system leveraging the potential of laser-based ablation for bio-sample preparation is proposed. It integrates and synchronizes all key components with extensive safety features and an intuitive user interface, allowing novice operators to perform sample preparations easily. As a first application, the proposed system has been utilized to create microdamages in mouse tail tendon fascicles. Promising results could be obtained, but careful tuning of the laser parameters and further optimization of the mechanical setup is still required to attain the high repeatability striven for

Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants.ISSN:2045-232

Biomaterial surface energy-driven ligand assembly strongly regulates stem cell mechanosensitivity and fate on very soft substrates

Although mechanisms of cell–material interaction and cellular mechanotransduction are increasingly understood, the mechanical insensitivity of mesenchymal cells to certain soft amorphous biomaterial substrates has remained largely unexplained. We reveal that surface energy-driven supramolecular ligand assembly can regulate mesenchymal stem cell (MSC) sensing of substrate mechanical compliance and subsequent cell fate. Human MSCs were cultured on collagen-coated hydrophobic polydimethylsiloxane (PDMS) and hydrophilic polyethylene-oxide-PDMS (PEO-PDMS) of a range of stiffnesses. Although cell contractility was similarly diminished on soft substrates of both types, cell spreading and osteogenic differentiation occurred only on soft PDMS and not hydrophilic PEO-PDMS (elastic modulus <1 kPa). Substrate surface energy yields distinct ligand topologies with accordingly distinct profiles of recruited transmembrane cell receptors and related focal adhesion signaling. These differences did not differentially regulate Rho-associated kinase activity, but nonetheless regulated both cell spreading and downstream differentiation.ISSN:0027-8424ISSN:1091-649

Inhibition of ERK 1/2 kinases prevents tendon matrix breakdown

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants