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Aging tendons undergo disruptions in homeostasis, increased susceptibility to injury, and reduced capacity for healing. Exploring the mechanisms behind this disruption in homeostasis is essential for developing therapeutics aimed at maintaining tendon health through the lifespan. We have previously identified that the extracellular matrix protein, Cochlin , which is highly expressed in healthy flexor tendon, is consistently lost during both natural aging and upon depletion of Scleraxis-lineage cells in young animals, which recapitulates many aging-associated homeostatic disruptions. Therefore, we hypothesized that loss of Cochlin would disrupt tendon homeostasis, including alterations in collagen fibril organization, and impaired tendon mechanics. By 3-months of age, Cochlin -/- flexor tendons exhibited altered collagen structure, with these changes persisting through at least 9-months. In addition, Cochlin -/- tendons demonstrated significant declines in structural and material properties at 6-months, and structural properties at 9-months. While Cochlin -/- did not drastically change the overall tendon proteome, consistent decreases in proteins associated with RNA metabolism, extracellular matrix production and the cytoskeleton were observed in Cochlin -/- . Interestingly, homeostatic disruption via Cochlin -/- did not impair the tendon healing process. Taken together, these data define a critical role for Cochlin in maintaining tendon homeostasis and suggest retention or restoration of Cochlin as a potential therapeutic approach to retain tendon structure and function through the lifespan.

Successful tendon healing requires sufficient deposition and remodeling of new extracellular matrix at the site of injury, with this process mediating in part through fibroblast activation via communication with macrophages. Moreover, resolution of healing requires clearance or reversion of activated cells, with chronic interactions with persistent macrophages impairing resolution and facilitating the conversion to fibrotic healing. As such, modulation of the macrophage environment represents an important translational target to improve the tendon healing process. Circulating monocytes are recruited to sites of tissue injury, including the tendon, via upregulation of cytokines including Ccl2, which facilitates recruitment of Ccr2 + macrophages to the healing tendon. Our prior work has demonstrated that Ccr2 -/- can modulate fibroblast activation and myofibroblast differentiation. However, this approach lacked temporal control and resulted in healing impairments. Thus, in the current study we have leveraged a Ccr2 antagonist to blunt macrophage recruitment to the healing tendon in a time-dependent manner. We first tested the effects of Ccr2 antagonism during the acute inflammatory phase and found that this had no effect on the healing process. In contrast, Ccr2 antagonism during the early proliferative/granulation tissue period resulted in significant improvements in mechanical properties of the healing tendon. Collectively, these data demonstrate the temporally distinct impacts of modulating Ccr2 + cell recruitment and Ccr2 antagonism during tendon healing and highlight the translational potential of transient Ccr2 antagonism to improve the tendon healing process., (© 2024 Orthopaedic Research Society.)

Tendon injuries disrupt successful transmission of force between muscle and bone, resulting in reduced mobility, increased pain, and significantly reduced quality of life for affected patients. There are currently no targeted treatments to improve tendon healing beyond conservative methods such as rest and physical therapy. Tissue engineering approaches hold great promise for designing instructive biomaterials that could improve tendon healing or for generating replacement graft tissue. More recently, engineered microphysiological systems to model tendon injuries have been used to identify therapeutic targets. Despite these advances, current tissue engineering efforts that aim to regenerate, replace, or model injured tendons have largely failed due in large part to a lack of understanding of how the mechanical environment of the tendon influences tissue homeostasis and how altered mechanical loading can promote or prevent disease progression. This review article draws inspiration from what is known about tendon loading from in vivo animal models and identifies key metrics that can be used to benchmark success in tissue engineering applications. Finally, we highlight important challenges and opportunities for the field of tendon tissue engineering that should be taken into consideration in designing engineered platforms to understand or improve tendon healing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Muscat and Nichols.)

Successful tendon healing requires sufficient deposition and remodeling of new extracellular matrix at the site of injury, with this process mediating in part through fibroblast activation via communication with macrophages. Moreover, resolution of healing requires clearance or reversion of activated cells, with chronic interactions with persistent macrophages impairing resolution and facilitating the conversion the conversion to fibrotic healing. As such, modulation of the macrophage environment represents an important translational target to improve the tendon healing process. Circulating monocytes are recruited to sites of tissue injury, including the tendon, via upregulation of cytokines including Ccl2, which facilitates recruitment of Ccr2+ macrophages to the healing tendon. Our prior work has demonstrated that Ccr2-/- can modulate fibroblast activation and myofibroblast differentiation. However, this approach lacked temporal control and resulted in healing impairments. Thus, in the current study we have leveraged a Ccr2 antagonist to blunt macrophage recruitment to the healing tendon in a time-dependent manner. We first tested the effects of Ccr2 antagonism during the acute inflammatory phase and found that this had no effect on the healing process. In contrast, Ccr2 antagonism during the late inflammatory/ early proliferative period resulted in significant improvements in mechanical properties of the healing tendon. Collectively, these data demonstrate the temporally distinct impacts of modulating Ccr2+ cell recruitment and Ccr2 antagonism during tendon healing and highlight the translational potential of transient Ccr2 antagonism to improve the tendon healing process.

Satisfactory healing following acute tendon injury is marred by fibrosis. Despite the high frequency of tendon injuries and poor outcomes, there are no pharmacological therapies in use to enhance the healing process. Moreover, systemic treatments demonstrate poor tendon homing, limiting the beneficial effects of potential tendon therapeutics. To address this unmet need, we leveraged our existing tendon healing spatial transcriptomics dataset and identified an area enriched for expression of Acp5 (TRAP) and subsequently demonstrated robust TRAP activity in the healing tendon. This unexpected finding allowed us to refine and apply our existing TRAP binding peptide (TBP) functionalized nanoparticle (NP) drug delivery system (DDS) to facilitate improved delivery of systemic treatments to the healing tendon. To demonstrate the translational potential of this DDS, we delivered niclosamide (NEN), an S100a4 inhibitor. While systemic delivery of free NEN did not alter healing, TBP-NP NEN enhanced both functional and mechanical recovery, demonstrating the translational potential of this approach to enhance the tendon healing process.

Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of αSMA+ stress fibers, αSMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of αSMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (Postn Lin ) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that Postn Lin cells contribute to a transient αSMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing., Competing Interests: Declaration of Competing Interest The authors declare that there are no competing interests with this manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Aged tendons have disrupted homeostasis, increased injury risk, and impaired healing capacity. Understanding mechanisms of homeostatic disruption is crucial for developing therapeutics to retain tendon health through the lifespan. Here, we developed a novel model of accelerated tendon extracellular matrix (ECM) aging via depletion of Scleraxis-lineage cells in young mice (Scx-DTR). Scx-DTR recapitulates many aspects of tendon aging including comparable declines in cellularity, alterations in ECM structure, organization, and composition. Single-cell RNA sequencing demonstrated a conserved decline in tenocytes associated with ECM biosynthesis in aged and Scx-DTR tendons, identifying the requirement for Scleraxis-lineage cells during homeostasis. However, the remaining cells in aged and Scx-DTR tendons demonstrate functional divergence. Aged tenocytes become pro-inflammatory and lose proteostasis. In contrast, tenocytes from Scx-DTR tendons demonstrate enhanced remodeling capacity. Collectively, this study defines Scx-DTR as a novel model of accelerated tendon ECM aging and identifies novel biological intervention points to maintain tendon function through the lifespan., Competing Interests: AK, AN, MB, AL No competing interests declared, (© 2023, Korcari et al.)

Tendon injuries heal via a scar-mediated response, and there are no biological approaches to promote more regenerative healing. Mouse flexor tendons heal through the formation of spatially distinct tissue areas: a highly aligned tissue bridge between the native tendon stubs that is enriched for adult Scleraxis-lineage cells and a disorganized outer shell associated with peri-tendinous scar formation. However, the specific molecular programs that underpin these spatially distinct tissue profiles are poorly defined. In the present study, we combine lineage tracing of adult Scleraxis-lineage cells with spatial transcriptomic profiling to define the overarching molecular programs that govern tendon healing and cell-fate decisions. Pseudotime analysis identified three fibroblast trajectories (synthetic, fibrotic, and reactive) and key transcription factors regulating these fate-switching decisions, including the progression of adult Scleraxis-lineage cells through the reactive trajectory. Collectively, this resource defines the molecular mechanisms that coordinate the temporo-spatial healing phenotype, which can be leveraged to inform therapeutic candidate selection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

During tendon healing, macrophages are thought to be a key mediator of scar tissue formation, which prevents successful functional restoration of the tendon. However, macrophages are critical for successful tendon healing as they aid in wound debridement, extracellular matrix deposition, and promote fibroblast proliferation. Recent work has sought to better define the multi-faceted functions of macrophages using depletion studies, while other studies have identified a tendon resident macrophage population. To begin to delineate the functions of tendon-resident versus circulation-derived macrophages, we examined the tendon healing phenotype in Chemokine Receptor 2 (CCR2) reporter (CCR2 GFP/+ ), and knockout mice. CCR2 is a chemokine receptor primarily found on the surface of circulating bone marrow-derived monocytes, with CCR2 being an important mediator of macrophage recruitment to wound environments. Surprisingly, CCR2 GFP/+ cells were present in the tendon during adult homeostasis, and single-cell RNA sequencing identified these cells as tendon-resident macrophages and T cells. During both homeostasis and healing, CCR2 knockout resulted in a substantial decrease in CCR2 GFP+ cells and pan-macrophages. Additionally, loss of CCR2 resulted in reduced numbers of myofibroblasts and impeded functional recovery during late healing. This study highlights the heterogeneity of tendon-resident and recruited immune cells and their contributions following injury, and establishes an important role for CCR2 in modulating both the adult tendon cell environment and tendon healing process., (© 2022 Federation of American Societies for Experimental Biology.)

The use of tamoxifen-inducible models of Cre recombinase in the tendon field is rapidly expanding, resulting in an enhanced understanding of tendon homeostasis and healing. However, the effects of tamoxifen on the tendon are not well-defined, which is particularly problematic given that tamoxifen can have both profibrotic and antifibrotic effects in a tissue-specific manner. Therefore, in the present study, we examined the effects of tamoxifen on tendon homeostasis and healing in male and female C57Bl/6J mice. Tamoxifen-treated mice were compared to corn oil (vehicle)-treated mice. In the "washout" treatment regimen, mice were treated with tamoxifen or corn oil for 3 days beginning 1 week prior to undergoing complete transection and surgical repair of the flexor digitorum longus tendon. In the second regimen, mice were treated with tamoxifen or corn oil beginning on the day of surgery, daily through day 2 postsurgery, and every 48 hours thereafter (D0-2q48) until harvest. All repaired tendons and uninjured contralateral control tendons were harvested at day 14 postsurgery. Tamoxifen treatment had no effect on tendon healing in male mice, regardless of the treatment regimen, while Max load was significantly decreased in female repairs in the Tamoxifen washout group, relative to corn oil. In contrast, D0-2q48 corn oil treatment in female mice led to substantial disruptions in tendon homeostasis, relative to washout corn oil treatment. Collectively, these data clearly define the functional effects of tamoxifen and corn oil treatment in the tendon and inform future use of tamoxifen-inducible genetic models., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Tendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell subpopulations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation methods preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior. Statement of clinical significance: The development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing., (© 2021 Federation of American Societies for Experimental Biology.)

Achilles tendinopathy is a debilitating condition affecting the entire spectrum of society and a condition that increases the risk of tendon rupture. Effective therapies remain elusive, as anti-inflammatory drugs and surgical interventions show poor long-term outcomes. Eccentric loading of the Achilles muscle-tendon unit is an effective physical therapy for treatment of symptomatic human tendinopathy. Here, we introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This model includes the application of tissue (muscle and tendon)-loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human clinical protocols. Under computer control, the foot was rotated through the entire ankle joint range of motion while the plantar flexors simultaneously contracted to simulate body mass loading, consistent with human therapeutic exercises. This approach achieved two key components of the heel drop and raise movement: ankle range of motion coupled with body mass loading. Model development entailed the tuning of parameters such as footplate speed, number of repetitions, number of sets of repetitions, treatment frequency, treatment duration, and treatment timing. Initial model development was carried out on uninjured mice to define a protocol that was well tolerated and nondeleterious to tendon biomechanical function. When applied to a murine Achilles tendinopathy model, muscle loading led to a significant improvement in biomechanical outcome measures, with a decrease in cross-sectional area and an increase in material properties, compared with untreated animals. Our model facilitates the future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries. NEW & NOTEWORTHY We introduce a novel mouse model of hindlimb muscle loading designed to achieve a tissue-targeted therapeutic exercise. This innovative model allows for application of muscle loading "doses," coupled with ankle dorsiflexion and plantarflexion, inspired by human loading clinical treatment. Our model facilitates future investigation of mechanisms whereby rehabilitative muscle loading promotes healing of Achilles tendon injuries.

Although inflammation is necessary during the early phases of tissue repair, persistent inflammation contributes to fibrosis. Acute tendon injuries often heal through a fibrotic mechanism, which impedes regeneration and functional recovery. Because inflammation mediated by nuclear factor κB (NF-κB) signaling is implicated in this process, we examined the spatial, temporal, and cell type-specific activation profile of canonical NF-κB signaling during tendon healing. NF-κB signaling was maintained through all phases of tendon healing in mice, including the remodeling phase, and tenocytes and myofibroblasts from the Scleraxis ( Scx ) lineage were the predominant populations that retained NF-κB activation into the late stages of repair. We confirmed persistent NF-κB activation in myofibroblasts in human tendon scar tissue. Deleting the canonical NF-κB kinase, IKKβ, in Scx -lineage cells in mice increased apoptosis and the deposition of the matrix protein periostin during the late stages of tendon repair, suggesting that persistent NF-κB signaling may facilitate myofibroblast survival and fibrotic progression. Consistent with this, myofibroblasts in human tendon scar samples displayed enhanced prosurvival signaling compared to control tissue. Together, these data suggest that NF-κB may contribute to fibrotic tendon healing through both inflammation-dependent and inflammation-independent functions, such as NF-κB-mediated cell survival., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Over 300,000 tendon repairs are performed annually in the United States to repair damage to tendons as a result of either acute trauma or chronic tendinopathy. Individuals with type II diabetes mellitus (T2DM) are four times more likely to experience tendinopathy, and up to five times more likely to experience a tendon tear or rupture than non-diabetics. As nearly 10% of the US population is diabetic, with an additional 33% pre-diabetic, this is a particularly problematic health care challenge. Tendon healing in general is challenging and often unsatisfactory due to the formation of mechanically inferior scar-tissue rather than regeneration of native tendon structure. In T2DM tendons, there is evidence of an amplified scar tissue response, which may be associated with the increased the risk of rupture or impaired restoration of range of motion. Despite the dramatic effect of T2DM on tendon function and outcomes following injury, there are few therapies available to promote improved healing in these patients. Several recent studies have enhanced our understanding of the pro-inflammatory environment of T2DM healing and have assessed potential treatment approaches to mitigate pathological progression in pre-clinical models of diabetic tendinopathy. This review discusses the current state of knowledge of diabetic tendon healing from molecular to mechanical disruptions and identifies promising approaches and critical knowledge gaps as the field moves toward identification of novel therapeutic strategies to maintain or restore tendon function in diabetic patients. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:13-22, 2020., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Extracellular matrix (ECM) is responsible for tendon strength and elasticity. Healed tendon ECM lacks structural integrity, leading to reinjury. Porcine urinary bladder matrix (UBM) provides a scaffold and source of bioactive proteins to improve tissue healing, but has received limited attention for treating tendon injuries. The objective of this study was to evaluate the ability of UBM to induce matrix organization and tenogenesis using a novel in vitro model. We hypothesized that addition of UBM to tendon ECM hydrogels would improve matrix organization and cell differentiation. Hydrogels seeded with bone marrow cells (n = 6 adult horses) were cast using rat tail tendon ECM ± UBM, fixed under static tension and harvested at 7 and 21 days for construct contraction, cell viability, histology, biochemistry, and gene expression. By day 7, UBM constructs contracted significantly from baseline, whereas control constructs did not. Both control and UBM constructs contracted significantly by day 21. In both groups, cells remained viable over time and changed from round and randomly oriented to elongated along lines of tension with visible compaction of the ECM. There were no differences over time or between treatments for nuclear aspect ratio, DNA, or glycosaminoglycan content. Decorin, matrix metalloproteinase 13, and scleraxis expression increased significantly over time, but not in response to UBM treatment. Mohawk expression was constant over time. Cartilage oligomeric matrix protein expression decreased over time in both groups. Using a novel ECM hydrogel model, substantial matrix organization and cell differentiation occurred; however, the addition of UBM failed to induce greater matrix organization than tendon ECM alone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1848-1859, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Tendon injuries are common and can dramatically impair patient mobility and productivity, resulting in a significant socioeconomic burden and reduced quality of life. Because the tendon healing process results in the formation of a fibrotic scar, injured tendons never regain the mechanical strength of the uninjured tendon, leading to frequent reinjury. Many tendons are also prone to the development of peritendinous adhesions and excess scar formation, which further reduce tendon function and lead to chronic complications. Despite this, there are currently no treatments that adequately improve the tendon healing process due in part to a lack of information regarding the contributions of various cell types to tendon healing and how their activity may be modulated for therapeutic value. In this review, we summarize recent efforts to identify and characterize the distinct cell populations involved at each stage of tendon healing. In addition, we examine the mechanisms through which different cell populations contribute to the fibrotic response to tendon injury, and how these responses can be affected by systemic factors and comorbidities. We then discuss gaps in our current understanding of tendon fibrosis and highlight how new technologies and research areas are shedding light on this clinically important and intractable challenge. A better understanding of the complex cellular environment during tendon healing is crucial to the development of new therapies to prevent fibrosis and promote tissue regeneration., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous population of cells that yields a homogeneous population of plastic-adherent adipose tissue-derived stromal cells (ASC) when culture-expanded. SVF and ASC have been used clinically to improve tendon healing, yet their mechanism of action is not fully elucidated. The objective of this study was to investigate the potential for ASC to act as trophic mediators for tendon healing. Flexor digitorum superficialis tendons and adipose tissue were harvested from adult horses to obtain SVF, ASC, and tenocytes. Growth factor gene expression was quantified in SVF and ASC in serial passages and growth factors were quantified in ASC-conditioned medium (CM). Microchemotaxis assays were performed using ASC-CM. Tenocytes were grown in co-culture with autologous ASC or allogeneic SVF. Gene expression for insulin-like growth factor 1 (IGF-1), stromal cell-derived factor-1α (SDF-1α), transforming growth factor-β1 (TGF-β1) and TGF-β3 was significantly higher in SVF compared to ASC. Concentrations were significantly increased in ASC-CM compared to controls for IGF-1 (4-fold) and SDF-1α (6-fold). Medium conditioned by ASC induced significant cell migration in a dose-dependent manner. Gene expression for collagen types I and III, decorin, and cartilage oligomeric matrix protein was modestly, but significantly increased following co-culture of tenocytes with autologous ASC. Our findings support the ability of SVF and ASC to act as trophic mediators in tendon healing, particularly through chemotaxis, which stands to critically impact the intrinsic healing response. In vivo studies to further delineate the potential for SVF and/or ASC to improve tendon healing are warranted. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1429-1439, 2019., (© 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Efforts to generate tissue-engineered anterior cruciate ligament replacements are limited by a lack of methods to derive mature ligament cells. Viral overexpression of the tendon/ligament marker scleraxis (Scx) can drive cell differentiation; however, the use of viral vectors hampers translation to clinical use. In this study, C3H10T1/2 cells were transiently transfected with expression vectors containing the full-length murine Scx cDNA and cultured in three-dimensional collagen hydrogels under static or cyclic strain for up to 14 days. β-galactosidase (LacZ) transfected cells served as controls. Cell morphology and gene expression for ligament-related genes, in addition to contraction (hydrogel width), mechanical properties, and glycosaminoglycan (GAG) and DNA content of hydrogels, were quantified and compared over time, between Scx and LacZ groups, and between static and cyclically strained constructs. Increased Scx expression was maintained for the entire 14-day study in both static and cyclically strained constructs. In static culture, overexpression of Scx resulted in greater cell elongation and construct contraction compared to LacZ controls. There were no differences in gene expression, DNA, or GAG content between Scx and LacZ constructs cultured under static conditions and no differences in DNA content between Scx and LacZ constructs. When exposed to cyclic strain, Scx-overexpressing cells maintained the elongated phenotype exhibited in static constructs, increased GAG production compared to static culture, and increased expression of the ligament-related genes collagen type I, decorin, and tenascin-C compared to strained LacZ controls. Cyclically strained constructs containing Scx-overexpressing cells had increased maximum load and stiffness compared to LacZ controls. The maintenance of increased Scx expression throughout the 14 day study and subsequent increases in ligament marker gene expression and mechanical properties with cyclic, but not static strain, suggest that transient transfection may be a viable alternative to viral transduction of Scx for ligament engineering studies and support a synergistic effect of Scx and mechanical strain on driving early ligament cell differentiation.

Background: Tendinopathies are common and difficult to resolve due to the formation of scar tissue that reduces the mechanical integrity of the tissue, leading to frequent reinjury. Tenocytes respond to both excessive loading and unloading by producing pro-inflammatory mediators, suggesting that these cells are actively involved in the development of tendon degeneration. The transcription factor scleraxis (Scx) is required for the development of force-transmitting tendon during development and for mechanically stimulated tenogenesis of stem cells, but its function in adult tenocytes is less well-defined. The aim of this study was to further define the role of Scx in mediating the adult tenocyte mechanoresponse., Results: Equine tenocytes exposed to siRNA targeting Scx or a control siRNA were maintained under cyclic mechanical strain before being submitted for RNA-seq analysis. Focal adhesions and extracellular matrix-receptor interaction were among the top gene networks downregulated in Scx knockdown tenocytes. Correspondingly, tenocytes exposed to Scx siRNA were significantly softer, with longer vinculin-containing focal adhesions, and an impaired ability to migrate on soft surfaces. Other pathways affected by Scx knockdown included increased oxidative phosphorylation and diseases caused by endoplasmic reticular stress, pointing to a larger role for Scx in maintaining tenocyte homeostasis., Conclusions: Our study identifies several novel roles for Scx in adult tenocytes, which suggest that Scx facilitates mechanosensing by regulating the expression of several mechanosensitive focal adhesion proteins. Furthermore, we identified a number of other pathways and targets affected by Scx knockdown that have the potential to elucidate the role that tenocytes may play in the development of degenerative tendinopathy.