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Inflammasomes are multiprotein complexes that regulate the bioactive production of IL-1β and IL-18, being implicated in the inflammatory response of different diseases. The inflammasome formed by the cytosolic sensor NLRP3 is highly promiscuous, as it could be activated by different pathogen- and sterile-signals. However, few models have studied the implication of NLRP3 in tissue damage-induced inflammation, particularly the implication of NLRP3 in tendinopathies. Here, we aimed to investigate the implication of NLRP3 in a mouse model of tendinopathy by collagenase degradation of the extracellular matrix in the Achilles' mice tendon. We found that NLRP3 was involved in the production of IL-1β, but another ASC-dependent inflammasome was required to produce IL-18 during sterile tissue damage. Our study suggests that in the immune response to extracellular matrix degradation different inflammasomes, probably expressed in different cell compartments, were able to differentially control IL-1β and IL-18 production in vivo. These results suggest the potential use of therapies targeting ASC as beneficial in the treatment of tendinopathies., (© 2024 The Author(s).)

Tendons play a crucial role in the musculoskeletal system, connecting muscles to bones and enabling efficient force transfer. However, they are prone to acute and chronic injuries, which, if not properly repaired, can significantly impair function. Tendinopathy, a prevalent condition affecting approximately 20% of musculoskeletal complaints, arises from an imbalance between micro-injury accumulation and repair processes. The extracellular matrix (ECM) of tendons is a hierarchical structure comprising collagen fibrils, proteoglycans, and glycoproteins that regulate organization, hydration, and mechanical properties. Mechanotransduction pathways, mediated by integrins and focal adhesion complexes, activate signaling cascades such as MAPK/ERK and PI3K/Akt, driving tenocyte gene expression and ECM remodeling. Adaptations to load involve region-specific remodeling, with tensile regions favoring aligned Type I collagen and compressive regions promoting proteoglycans like aggrecan. Stress shielding or reduced loading disrupts these pathways, leading to matrix disorganization and inflammation, predisposing tendons to degenerative changes. Insights into these molecular mechanisms inform rehabilitation strategies to enhance tendon repair and mitigate tendinopathy progression in both athletic and general populations., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Objective: To assess the mechanism of supraspinatus tendinopathy induced by type 2 diabetes mellitus (T2DM) in rats using untargeted metabolomics analysis., Methods: The liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics approach was used to screen tendon biomarkers of supraspinatus tendinopathy in rats with T2DM. Seventy-eight Sprague-Dawley rats were divided into normal group (NG) and T2DM groups. Rats in T2DM groups were divided into 12-week (T2DM-12w), and 24-week (T2DM-24w) subgroups according to the time point of the establishment of the T2DM rat model. Histological evaluation (modified Bonar score) and biomechanical testing were used to analyze the adverse effects of type 2 diabetes on the tendon of the supraspinatus muscle in rats.Three comparable groups were set up, including T2DM-12w group vs. NG, T2DM-24w group vs. NG, and T2DM-24w group vs. T2DM-12w group. Differentially expressed metabolites (DEMs) in the supraspinatus tendons in the three groups of rats were analyzed using LC-MS, and data were analyzed using multivariate statistical methods to screen potential biomarkers. The DEMs included in the intersection of the three groups were identified as those associated with the development of diabetic supraspinatus tendinopathy, and trend analysis and pathway topology analysis were performed., Results: With the progression of diabetes, the tendinopathy of the supracinatus muscle of diabetic rats gradually intensified, mainly manifested as inflammatory reactions, disordered collagen fibers, fat infiltration, and increased modified Bonar score. The intersection of DEMs among the three comparable groups was resulted in the identification of 10 key DEMs, in which melezitose and raffinose showed a continuous increasing trend with the prolongation of disease course. By pathway topology analysis, 10 DEMs (P < 0.01) were mainly associated with the pathways of galactose metabolism, which could be involved in the development of diabetes-induced supraspinatus tendinopathy., Conclusion: T2DM causes tendinopathy of the supraspinatus muscle in rats. 10 key DEMs obtained by untargeted metabolomics assay suggested that the development of diabetes-induced supraspinatus tendinopathy was associated with changes in metabolic pathways, such as galactose metabolism. melezitose and raffinose hold promise as a biomarker for disease discrimination and/or disease indication in diabetic supraspinatus tendinopathy., Competing Interests: Declarations Ethics approval and consent to participate This study was carried out in accordance with NIH guidelines for the care and use of laboratory animals (8th edition, NIH). The study protocol was approved by the Ethics Committee of Experimental Animals of the Affiliated Hospital of Qingdao University (Approval No. 20220505SD8020221210126). All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The lack of representative in vitro models recapitulating human tendon (patho)physiology is among the major factors hindering consistent progress in the knowledge-based development of adequate therapies for tendinopathy.Here, an organotypic 3D tendon-on-chip model is designed that allows studying the spatiotemporal dynamics of its cellular and molecular mechanisms.Combining the synergistic effects of a bioactive hydrogel matrix with the biophysical cues of magnetic microfibers directly aligned on the microfluidic chip, it is possible to recreate the anisotropic architecture, cell patterns, and phenotype of tendon intrinsic (core) compartment. When incorporated with vascular-like vessels emulating the interface between its intrinsic-extrinsic compartments, crosstalk with endothelial cells are found to drive stromal tenocytes toward a reparative profile. This platform is further used to study adaptive immune cell responses at the onset of tissue inflammation, focusing on interactions between tendon compartment tenocytes and circulating T cells.The proinflammatory signature resulting from this intra/inter-cellular communication induces the recruitment of T cells into the inflamed core compartment and confirms the involvement of this cellular crosstalk in positive feedback loops leading to the amplification of tendon inflammation.Overall, the developed 3D tendon-on-chip provides a powerful new tool enabling mechanistic studies on the pathogenesis of tendinopathy as well as for assessing new therapies., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Tendon stem/progenitor cells (TSPCs) are crucial for tendon repair, regeneration, and homeostasis. Dysfunction of TSPCs, due to aberrant activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, contributes to tendinopathy. Unfortunately, the effectiveness of conventional subcutaneous injection targeting at suppressing JAK/STAT signaling pathway is limited due to the passive diffusion of drugs away from the injury site. Herein, a novel poly-gamma-glutamic acid (γ-PGA) dual-barb microneedle (MN) path loaded with TSPCs-derived nanovesicles (NVs) containing JAK/STAT inhibitor WP1066 (MN-WP1066-NVs) for tendinopathy treatment is designed. The dual-barb design of the MN ensures firm adhesion to the skin, allowing for sustained and prolonged release of WP1066-NVs, facilitating enhanced TSPCs self-renewal, migration, and stemness in tendinopathy. In vitro and in vivo experiments demonstrate that the degradation of γ-PGA patch tips facilitates the gradual release of WP1066-NVs at the lesion site. This release alleviates inflammation, suppresses extracellular matrix degradation, and restores normal tendon histological structure by inhibiting the JAK/STAT pathway. These findings suggest that the multifunctional dual-barb MN patch offers a novel and effective therapeutic strategy for tendinopathy treatment., (© 2024 Wiley‐VCH GmbH.)

Background: Tendinopathy alters the compositional properties of the Achilles tendon by increasing fluid and glycosaminoglycan content. It has been speculated that these changes may affect intratendinous pressure, but the extent of this relationship remains unclear. Therefore, we aimed to investigate the impact of elevated fluid and glycosaminoglycan content on Achilles tendon intratendinous pressure and to determine whether hyaluronidase (HYAL) therapy can intervene in this potential relationship., Methods: Twenty paired fresh-frozen cadaveric Achilles tendons were mounted in a tensile-testing machine and loaded up to 5% strain. Intratendinous resting (at 0% strain) and dynamic pressure (at 5% strain) were assessed using the microcapillary infusion technique. First, intratendinous pressure was measured under native conditions before and after infusion of 2 mL physiological saline. Next, 80 mg of glycosaminoglycans were administered bilaterally to the paired tendons. The right tendons were additionally treated with 1500 units of HYAL. Finally, both groups were retested, and the glycosaminoglycan content was analyzed., Results: It was found that both elevated fluid and glycosaminoglycan content resulted in higher intratendinous resting and dynamic pressures (p < 0.001). HYAL treatment induced a 2.3-fold reduction in glycosaminoglycan content (p = 0.002) and restored intratendinous pressures., Conclusion: The results of this study demonstrated that elevated fluid and glycosaminoglycan content in Achilles tendinopathy contribute to increased intratendinous resting and dynamic pressures, which can be explained by the associated increased volume and reduced permeability of the tendon matrix, respectively. HYAL degrades glycosaminoglycans sufficiently to lower intratendinous pressures and may, therefore, serve as a promising treatment., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Achilles tendinopathy is often attributed to overuse, but its pathophysiology remains poorly understood. Disruption to the molecular structure of collagen is fundamental for the onset and progression of tendinopathy but has mostly been investigated in vitro. Here, we interrogated the in vivo molecular structure changes of collagen in rat Achilles tendons following treadmill running. Unexpectedly, the tendons' collagen molecules were not mechanically unfolded by running but denatured through proteolysis during physiological post-run remodeling. We further revealed that running induces inflammatory gene expressions in Achilles tendons and that long-term running causes prolonged, elevated collagen degradation, leading to the accumulation of denatured collagen and tendinopathy development. For applications, we demonstrated magnetic resonance imaging of collagenase-induced Achilles tendon injury in vivo using a denatured collagen targeting contrast agent. Our findings may help close the knowledge gaps in the mechanobiology and pathogenesis of Achilles tendinopathy and initiate new strategies for its imaging-based diagnosis.

Purpose: This study aims to assess ultrashort-TE magnetization transfer (UTE-MT) imaging of collagen degradation using an in vitro model of rotator cuff tendinopathy., Methods: Thirty-six supraspinatus tendon specimens were divided into three groups and treated with 600 U collagenase (Group 1), 150 U collagenase (Group 2), and phosphate buffer saline (Group 3). UTE-MT imaging was performed to assess changes in macromolecular fraction (MMF), macromolecule transverse relaxation time (T 2m ), water longitudinal relaxation rate constant (R 1m ), the magnetization exchange rate from the macromolecular to water pool (Rm 0 w ) and from water to the macromolecular pool (Rm 0 m ), and magnetization transfer ratio (MTR) at baseline and following digestion and their differences between groups. Biochemical and histological studies were conducted to determine the extent of collagen degradation. Correlation analyses were performed with MMF, T 2m , R 1m , Rm 0 w , Rm 0 m , and MTR, respectively. Univariate and multivariate linear regression analyses were performed to evaluate combinations of UTE-MT parameters to predict collagen degradation., Results: MMF, T 2m , R 1m , Rm 0 m , and MTR decreased after digestion. MMF (r = -0.842, p < 0.001), MTR (r = -0.78, p < 0.001), and Rm 0 m (r = -0.662, p < 0.001) were strongly negatively correlated with collagen degradation. The linear regression model of differences in MMF and Rm 0 m before and after digestion explained 68.9% of collagen degradation variation in the tendon. The model of postdigestion in MMF and T 2m and the model of MTR explained 54.2% and 52.3% of collagen degradation variation, respectively., Conclusion: This study highlighted the potential of UTE-MT parameters for evaluation of supraspinatus tendinopathy., (© 2024 International Society for Magnetic Resonance in Medicine.)

Tendinopathy is one of the most prevalent sports injury diseases in orthopedics. However, there is no effective treatment or medicine. Recently, the discovery of tendon stem cells (TSCs) provides a new perspective to find new therapeutic methods for Tendinopathy. Studies have shown that oxidative stress will inevitably cause TSCs injury during tendinopathy, but the mechanism has not been fully elucidated. Here, we report the oxidative damage of TSCs induced by H 2 O 2 via ferroptosis, as well, treatment with H 2 O 2 raised the proportion of mitochondria engulfed by autophagosomes in TSCs. The suppression of mitophagy by Mdivi-1 significantly attenuates the H 2 O 2 -induced ferroptosis in TSCs. Mechanically, H 2 O 2 actives the cGAS-STING pathway, which can regulate the level of mitophagy. Interfering with cGAS could impair mitophagy and the classical ferroptotic events. In the rat model of tendinopathy, interference of cGAS could relieve tendon injury by inhibiting ferroptosis. Overall, these results provided novel implications to reveal the molecular mechanism of tendinopathy, by which pointed to cGAS as a potential therapeutic target for the treatment of tendinopathy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells ( h TSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-β1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased h TSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased h TSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

Advanced age is a major risk factor for age-related degenerative tendinopathy. During aging, tendon stem/progenitor cell (TSPC) function declines owing to the transition from a normal quiescent state to a senescent state. Extracellular vesicles (EVs) from young stem cells are reported to possess anti-aging functions. However, it remains unclear whether EVs from young TSPCs (TSPC-EVs) can rejuvenate senescent TSPCs to delay age-related degeneration. Here, this study finds that TSPC-EVs can mitigate the aging phenotypes of senescent TSPCs and maintain their tenogenic capacity. In vitro studies reveal that TSPC-EVs can reinstall autophagy in senescent TSPCs to alleviate cellular senescence, and that the re-establishment of autophagy is mediated by the PI3K/AKT pathway. Mechanistically, this study finds that thrombospondin 1, a negative regulator of the PI3K/AKT pathway, is enriched in TSPC-EVs and can be transported to senescent TSPCs. Moreover, in vivo studies show that the local delivery of TSPC-EVs can rejuvenate senescent TSPCs and promote their tenogenic differentiation, thereby rescuing tendon regeneration in aged rats. Taken together, TSPC-EVs as a novel cell-free approach have promising therapeutic potential for aging-related degenerative tendinopathy., (© 2024 Wiley‐VCH GmbH.)

This study was designed to investigate the accumulation of advanced glycation end-products (AGEs) and the expression of the receptor of AGEs (RAGE) in tendinopathic tissues. In this study, tendinopathic posterior tibial tendons (PTT) were collected from patients (n=6). Redundant autografts of flexor digitorum longus tendon (FDL; n=3) were used for controls. The control and tendinopathic tendon tissues were used for extraction of proteins for western blot and sectioned for histology and immunohistochemistry. Tendinopathy of the PTT was confirmed histologically by the presentation of disorderly organized collagen fibers, high cellularity and increased vascularity. By immunohistochemistry, heterogeneous accumulation of AGEs was detected on the PTT sections and concentrated in areas, where collagen fibers were disorderly and tangled. In the PTT, roundish tenocytes were also AGEs-positive. In contrast, AGEs were diffuse, lightly stained in the FDL. A greater number of tenocytes within the tendinopathic lesions in the PTT were RAGE positive, compared to the tenocytes in the FDL. Western blot confirmed the expression of AGEs and RAGE in both tendinopathic PTT and control FDL but their band densities were not significantly different. The spatial relation of the accumulated AGEs and RAGE- positive tenocytes within the tendinopathic lesions indicates their involvement in the molecular pathology of tendinopathy., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Tendinopathy is a common age-related disease which causes significant morbidity for both human athletes and performance horses. In the latter, the superficial digital flexor tendon is an excellent model for human tendinopathies because it is a functional homologue of the human Achilles tendon and a primary site of injuries with strong similarities to the human disease. Corticosteroids have been previously used clinically to treat tendinopathic inflammation, but they upregulate the p53-p21 axis with concomitant reductions in cell proliferation and collagen synthesis in human tenocytes. This phenotype is consistent with the induction of cellular senescence in vitro and in vivo and probably represents an important clinical barrier to their effective use. Because of the many differences in senescence mechanisms between species, this study aimed to evaluate these mechanisms after corticosteroid treatment in equine tenocytes. Exposure to clinically reflective levels of dexamethasone for 48 hours drove equine tenocytes into steroid induced senescence (SIS). This was characterised by permanent growth arrest and upregulation of p53, the cyclin dependent kinase inhibitors p21waf and p16ink4a as well as the matrix degrading enzymes MMP1, MMP2 and MMP13. SIS also induced a distinctive equine senescence associated secretory phenotype (eSASP) characterised by enhanced secretion of IL-8 and MCP-1. Preincubation with resveratrol or the potent SIRT1 activator SRT1720 prevented SIS in equine tenocytes, while treatment with the non-SIRT1 activating resveratrol analogue V29 was equally protective against SIS, consistent with a novel, as yet uncharacterised SIRT1-indendent mechanism which has relevance for the development of future preventative and therapeutic strategies., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Heidari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tendon disorders often result in decreased muscle function and atrophy. Pulsed Electromagnetic Fields (PEMFs) have shown potential in improving tendon fiber structure and muscle recovery. However, the molecular effects of PEMF therapy on skeletal muscle, beyond conventional metrics like MRI or markers of muscle decline, remain largely unexplored. This study investigates the metabolic and structural changes in PEMF-treated muscle tissue using proteomics in a rat model of Achilles tendinopathy induced by collagenase. Sprague Dawley rats were unilaterally induced for tendinopathy with type I collagenase injection and exposed to PEMFs for 8 h/day. Gastrocnemius extracts from untreated or PEMF-treated rats were analyzed with LC-MS/MS, and proteomics differential analysis was conducted through label-free quantitation. PEMF-treated animals exhibited decreased glycolysis and increased LDHB expression, enhancing NAD signaling and ATP production, which boosted respiratory chain activity and fatty acid beta-oxidation. Antioxidant protein levels increased, controlling ROS production. PEMF therapy restored PGC1alpha and YAP levels, decreased by tendinopathy. Additionally, myosins regulating slow-twitch fibers and proteins involved in fiber alignment and force transmission increased, supporting muscle recovery and contractile function. Our findings show that PEMF treatment modulates NAD signaling and oxidative phosphorylation, aiding muscle recovery through the upregulation of YAP and PGC1alpha and increasing slow myosin isoforms, thus speeding up physiological recovery.

Tendinopathy is one of the most frequent musculoskeletal disorders characterized by sustained tissue inflammation and oxidative stress, accompanied by extracellular matrix remodeling. Patients suffering from this pathology frequently experience pain, swelling, stiffness, and muscle weakness. Current pharmacological interventions are based on nonsteroidal anti-inflammatory drugs; however, the effectiveness of these strategies remains ambiguous. Accumulating evidence supports that oral supplementation of natural compounds can provide preventive, and possibly curative, effects. Vitamin C (Vit C), collagen peptides (Coll), resveratrol (Res), and astaxanthin (Asx) were reported to be endowed with potential beneficial effects based on their anti-inflammatory and antioxidant activities. Here, we analyzed the efficacy of a novel combination of these compounds (Mix) in counteracting proinflammatory (IL-1 β ) and prooxidant (H 2 O 2 ) stimuli in human tenocytes. We demonstrated that Mix significantly impairs IL-6-induced IL-1 β secretion, NF- κ B nuclear translocation, and MMP-2 production; notably, a synergistic effect of Mix over the single compounds could be observed. Moreover, Mix was able to significantly counteract H 2 O 2 -triggered ROS production. Together, these results point out that Mix, a novel combination of Vit C, Coll, Resv, and Asx, significantly impairs proinflammatory and prooxidant stimuli in tenocytes, mechanisms that contribute to the onset of tendinopathies., Competing Interests: Dr. Stefania Battaglia, Dr. Marco Cozzi, Dr. Francesca R. Ranieri, and Dr. Valeria Curti are employees of Kolinpharma S.p.A., a nutraceutical company that produces and commercializes food supplements. These authors report no involvement of the company in the research that could have influenced the outcome of this work. The other authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2024 Monica Marzagalli et al.)

Achilles tendinopathy (TP) is characterized as the third most common disease of the musculoskeletal system, and occurs in three phases. There is currently no evidence of effective treatment for this medical condition. In this study, the modulatory effects of the minimally invasive technique intratissue percutaneous electrolysis (EPI) and combinations of EPI with four nutritional factors included in the diet, hydroxytyrosol (HT), maslinic acid (MA), glycine, and aspartate (AA), on hepatic intermediary metabolism was examined in Wistar rats with induced tendinopathy at various stages of TP. Results obtained showed that induced tendinopathy produced alterations in the liver intermediary metabolisms of the rats. Regarding carbohydrate metabolism, a reduction in the activity of pro-inflammatory enzymes in the later stages of TP was observed following treatment with EPI alone. Among the combined treatments using nutritional factors with EPI, HT+EPI and AA+EPI had the greatest effect on reducing inflammation in the late stages of TP. In terms of lipid metabolism, the HT+EPI and AA+EPI groups showed a decrease in lipogenesis. In protein metabolism, the HT+EPI group more effectively reduced the inflammatory effects of induced TP. Treatment with EPI combined with nutritional factors might help regulate intermediary metabolism in TP disease and reduce the inflammation process.

Both osteoporosis and tendinopathy are widely prevalent disorders, encountered in diverse medical contexts. Whilst each condition has distinct pathophysiological characteristics, they share several risk factors and underlying causes. Notably, oxidative stress emerges as a crucial intersecting factor, playing a pivotal role in the onset and progression of both diseases. This imbalance arises from a dysregulation in generating and neutralising reactive oxygen species (ROS), leading to an abnormal oxidative environment. Elevated levels of ROS can induce multiple cellular disruptions, such as cytotoxicity, apoptosis activation and reduced cell function, contributing to tissue deterioration and weakening the structural integrity of bones and tendons. Antioxidants are substances that can prevent or slow down the oxidation process, including Vitamin C, melatonin, resveratrol, anthocyanins and so on, demonstrating potential in treating these overlapping disorders. This comprehensive review aims to elucidate the complex role of oxidative stress within the interlinked pathways of these comorbid conditions. By integrating contemporary research and empirical findings, our objective is to outline new conceptual models and innovative treatment strategies for effectively managing these prevalent diseases. This review underscores the importance of further in-depth research to validate the efficacy of antioxidants and traditional Chinese medicine in treatment plans, as well as to explore targeted interventions focused on oxidative stress as promising areas for future medical advancements., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Mast cells are immune cells minimally present in normal tendon tissue. The increased abundance of mast cells in tendinopathy biopsies and at the sites of tendon injury suggests an unexplored role of this cell population in overuse tendon injuries. Mast cells are particularly present in tendon biopsies from patients with more chronic symptom duration and a history of intensive mechanical loading. This study, therefore, examined the cross talk between mast cells and human tendon cells in either static or mechanically active conditions in order to explore the potential mechanistic roles of mast cells in overuse tendon injuries. A coculture of isolated human tenocytes and mast cells (HMC-1) combined with Flexcell Tension System for cyclic stretching of tenocytes was used. Additionally, human tenocytes were exposed to agonists and antagonists of substance P (SP) receptors. Mast cell degranulation was assessed by measuring β-hexosaminidase activity. Transwell and cell adhesion assays were used to evaluate mast cell migration and binding to tendon extracellular matrix components (collagen and fibronectin), respectively. Gene expressions were analyzed using real time qRT-PCR. Our results indicate that mechanical stimulation of human tenocytes leads to release of SP which, in turn, activates mast cells through the Mas-related G-protein-coupled receptor X2 (MRGPRX2). The degranulation and migration of mast cells in response to MRGPRX2 activation subsequently cause human tenocytes to increase their expression of inflammatory factors, matrix proteins and matrix metalloproteinase enzymes. These observations may be important in understanding the mechanisms by which tendons become tendinopathic in response to repetitive mechanical stimulation., (© 2024. The Author(s).)

Background: Plantar fasciitis is a painful tendinous condition (tendinopathy) with a high prevalence in athletes. While a healthy tendon has limited blood flow, ultrasound has indicated elevated blood flow in tendinopathy, but it is unknown if this is related to a de facto increase in the tendon vasculature. Likewise, an accumulation of glycosaminoglycans (GAGs) is observed in tendinopathy, but its relationship to clinical pain is unknown., Purpose: To explore to what extent vascularization, inflammation, and fat infiltration were present in patients with plantar fasciitis and if they were related to clinical symptoms., Study Design: Descriptive laboratory study., Methods: Biopsy specimens from tendinopathic plantar fascia tissue were obtained per-operatively from both the primary site of tendon pain and tissue swelling ("proximal") and a region that appeared macroscopically healthy at 1 to 2 cm away from the primary site ("distal") in 22 patients. Biopsy specimens were examined with immunofluorescence for markers of blood vessels, tissue cell density, fat infiltration, and macrophage level. In addition, pain during the first step in the morning (registered during an earlier study) was correlated with the content of collagen and GAGs in tissue., Results: High vascularization (and cellularity) was present in both the proximal (0.89%) and the distal (0.96%) plantar fascia samples, whereas inconsistent but not significantly different fat infiltration and macrophage levels were observed. The collagen content was similar in the 2 plantar fascia regions, whereas the GAG content was higher in the proximal region (3.2% in proximal and 2.8% in distal; P = .027). The GAG content in the proximal region was positively correlated with the subjective morning pain score in the patients with tendinopathy (n = 17)., Conclusion: In patients with plantar fasciitis, marked tissue vascularization was present in both the painful focal region and a neighboring nonsymptomatic area. In contrast, the accumulation of hydrophilic GAGs was greater in the symptomatic region and was positively correlated with increased clinical pain levels in daily life., Clinical Relevance: The accumulation of GAGs in tissue rather than the extent of vascularization appears to be linked with the clinical degree of pain symptoms of the disease., Competing Interests: The authors declared that they have no conflicts of interest in the authorship and publication of this contribution. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

The effect of interleukin-38 (IL-38), a recently identified member of the IL-1 family with potential applications in various inflammation-related conditions, on ER stress has not been explored. Furthermore, its role in obesity-associated tendinopathy has not been investigated. In this study, human primary tenocytes were treated with palmitate (200 or 400 μM) and palmitate plus IL-38 (0-50 ng/mL) for 24 h. Western blotting was used to assess ER stress and tendinopathogenic markers in tenocytes. Monodansylcadaverine (MDC) staining was used to evaluate autophagosomes. Apoptosis was determined by cell viability assays, caspase 3 activity assays and TUNEL assays. Cell migration was evaluated by a cell scratch assay. Small interfering (si) RNA transfection was used for target gene silencing. Treatment of tenocytes with IL-38 attenuated apoptosis, restored the balance between MMPs and TIMP-1, and alleviated ER stress under palmitate conditions. IL-38 treatment enhanced AMPK phosphorylation and promoted the expression of autophagy markers related to LC3 conversion, p62 degradation, and autophagosome formation in cultured tenocytes. The effects of IL-38 on ER stress, apoptosis, and MMP-9, MMP-13, and TIMP-1 expression in palmitate-treated tenocytes were abrogated by AMPK siRNA or 3-methyladenine (3MA). These results suggest that IL-38 alleviates ER stress through the AMPK/autophagy pathway, thereby reducing apoptosis and preventing extracellular matrix (ECM) degradation in tenocytes under hyperlipidemic conditions. This study provides a promising therapeutic avenue for treating obesity-related tendinopathy using an endogenous compound such as IL-38., Competing Interests: Conflicts of Interest None, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tendinopathy, characterized by inflammatory and degenerative changes, presents challenges in sports and medicine. In addressing the limitations of conservative management, this study focuses on developing tendon grafts using extrusion bioprinting with platelet-rich plasma (PRP)-infused hydrogels loaded with tendon cells. The objective is to understand paracrine interactions initiated by bioprinted tendon grafts in either inflamed or non-inflamed host tissues. PRP was utilized to functionalize methacrylate gelatin (GelMA), incorporating tendon cells for graft bioprinting. Bioinformatic analyses of overexpressed proteins, predictive of functional enrichment, revealed insights into PRP graft behavior in both non-inflamed and inflamed environments. PRP grafts activated inflammatory pathways, including Interleukin 17 (IL-17), neuroinflammation, Interleukin 33 (IL-33), and chemokine signaling. Interleukin 1 beta (IL-1b) in the graft environment triggered p38 mitogen-activated protein kinase (MAPK) signaling, nuclear factor kappa light chain enhancer of activated B cells (NF-kB) canonical pathway, and Vascular Endothelial Growth Factor (VEGF) signaling. Biological enrichment attributed to PRP grafts included cell chemotaxis, collagen turnover, cell migration, and angiogenesis. Acellular PRP grafts differed from nude grafts in promoting vessel length, vessel area, and junction density. Angiogenesis in cellular grafts was enhanced with newly synthesized Interleukin 8 (IL-8) in cooperation with IL-1b. In conclusion, paracrine signaling from PRP grafts, mediated by chemokine activities, influences cell migration, inflammation, and angiogenic status in host tissues. Under inflammatory conditions, newly synthesized IL-8 regulates vascularization in collaboration with PRP.

Rotator cuff repair is usually successful, but retear is not uncommon. It has been previously identified that there is a higher incidence of apoptosis in the edges of the torn supraspinatus tendon. A prospective cohort study was conducted with 28 patients-14 rotator cuff tear patients, 5 instability patients, and 9 Anterior cruciate ligament reconstruction patients to determine whether there was any increase in several genes implicated in apoptosis, including Fas receptor (FasR), Fas ligand, Aifm-1, Bcl-2, Fadd, Bax, and caspase-3. There was a significant expression of Bax (P=0.2) and FasR (P=0.005) in the edges of torn supraspinatus tendons, and in intact subscapularis tendons, there was a significant expression of caspase-3 (P=0.02) compared with samples from the torn supraspinatus tendon (P=0.04). The cytochrome c pathway, with its subsequent activation of caspase-3, as well as the TRAIL-receptor signaling pathway involving FasR have both been implicated. The elevated expression of Bax supported the model that the Bax to Bcl-2 expression ratio represents a cell death switch. The elevated expression of Bax in the intact subscapularis tissue from rotator cuff tear patients also may confirm that tendinopathy is an ongoing molecular process., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Patellar tendinopathy is a common clinical problem, but its underlying pathophysiology remains poorly understood, primarily due to the absence of a representative experimental model. The most widely used method to generate such a model is collagenase injection, although this method possesses limitations. We developed an optimized rat model of patellar tendinopathy via the ultrasound-guided injection of collagenase mixed with a thermo-responsive Pluronic hydrogel into the patellar tendon of sixty male Wistar rats. All analyses were carried out at 3, 7, 14, 30, and 60 days post-injury. We confirmed that our rat model reproduced the pathophysiology observed in human patients through analyses of ultrasonography, histology, immunofluorescence, and biomechanical parameters. Tendons that were injured by the injection of the collagenase-Pluronic mixture exhibited a significant increase in the cross-sectional area ( p < 0.01), a high degree of tissue disorganization and hypercellularity, significantly strong neovascularization ( p < 0.01), important changes in the levels of types I and III collagen expression, and the organization and presence of intra-tendinous calcifications. Decreases in the maximum rupture force and stiffness were also observed. These results demonstrate that our model replicates the key features observed in human patellar tendinopathy. Collagenase is evenly distributed, as the Pluronic hydrogel prevents its leakage and thus, damage to surrounding tissues. Therefore, this model is valuable for testing new treatments for patellar tendinopathy.

The prevalence of tendinopathy in patients with diabetes is well documented. Despite efforts to improve diabetes management, there is a lack of research on therapeutic agents targeting the core features of tendinopathy, namely, tenocyte apoptosis and extracellular matrix (ECM) damage. In this study, we investigated the potential of ginsenoside compound K (CK), known for its antidiabetic properties, to mitigate tenocyte apoptosis, inflammation, oxidative stress, and the metalloproteinase (MMP) system under hyperglycemic conditions. Our research also aimed to unravel the molecular mechanism underlying the effects of CK. The assessment of apoptosis involved observing intracellular chromatin condensation and measuring caspase 3 activity. To gauge oxidative stress, we examined cellular ROS levels and hydrogen peroxide and malondialdehyde concentrations. Western blotting was employed to determine the expression of various proteins. Our findings indicate that CK treatment effectively countered high glucose-induced apoptosis, inflammation, and oxidative stress in cultured tenocytes. Furthermore, CK normalized the expression of MMP-9, MMP-13, and TIMP-1. Notably, CK treatment boosted the expression of PPARγ and antioxidant enzymes. We conducted small interfering (si) RNA experiments targeting PPARγ, revealing its role in mediating CK's effects on tendinopathy features in hyperglycemic tenocytes. In conclusion, these in vitro results offer valuable insights into the potential therapeutic role of CK in managing tendinopathy among individuals with diabetes. By addressing crucial aspects of tendinopathy, CK presents itself as a promising avenue for future research and treatment development in this domain., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Tendinopathy is a disorder of musculoskeletal system that primarily affects athletes and the elderly. Current treatment options are generally comprised of various exercise and loading programs, therapeutic modalities, and surgical interventions and are limited to pain management. This study is to understand the role of TRIM54 (tripartite motif containing 54) in tendonitis through in vitro modeling with tendon-derived stem cells (TDSCs) and in vivo using rat tendon injury model. Initially, we observed that TRIM54 overexpression in TDSCs model increased stemness and decreased apoptosis. Additionally, it rescued cells from tumor necrosis factor α-induced inflammation, migration, and tenogenic differentiation. Further, through immunoprecipitation studies, we identified that TRIM54 regulates inflammation in TDSCs by binding to and ubiquitinating YOD1. Further, overexpression of TRIM54 improved the histopathological score of tendon injury as well as the failure load, stiffness, and young modulus in vivo. These results indicated that TRIM54 played a critical role in reducing the effects of tendon injury. Consequently, these results shed light on potential therapeutic alternatives for treating tendinopathy., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Because equine tendinopathies are slow to heal and often recur, therapeutic strategies are being considered that aid tendon repair. Given the success of utilizing vitamin C to promote tenogenesis in other species, we hypothesized that vitamin C supplementation would produce dose-dependent improvements in the tenogenic properties of tendon proper (TP) and peritenon (PERI) cells of the equine superficial digital flexor tendon (SDFT). Equine TP- and PERI-progenitor-cell-seeded fibrin three-dimensional constructs were supplemented with four concentrations of vitamin C. The gene expression profiles of the constructs were assessed with 3'-Tag-Seq and real-time quantitative polymerase chain reaction (RT-qPCR); collagen content and fibril ultrastructure were also analyzed. Moreover, cells were challenged with dexamethasone to determine the levels of cytoprotection afforded by vitamin C. Expression profiling demonstrated that vitamin C had an anti-inflammatory effect on TP and PERI cell constructs. Moreover, vitamin C supplementation mitigated the degenerative pathways seen in tendinopathy and increased collagen content in tendon constructs. When challenged with dexamethasone in two-dimensional culture, vitamin C had a cytoprotective effect for TP cells but not necessarily for PERI cells. Future studies will explore the effects of vitamin C on these cells during inflammation and within the tendon niche in vivo.

Tendinopathy is one of the most common musculoskeletal disorders with significant repercussions on quality of life and sport activities. Physical exercise (PE) is considered the first-line approach to treat tendinopathy due renowned mechanobiological effects on tenocytes. Irisin, a recently identified myokine released during PE, has been recognized for several beneficial effects towards muscle, cartilage, bone, and intervertebral disc tissues. The aim of this study was to evaluate the effects of irisin on human primary tenocytes (hTCs) in vitro. Human tendons were harvested from specimens of patients undergoing anterior cruciate ligament reconstruction (n = 4). After isolation and expansion, hTCs were treated with RPMI medium (negative control), interleukin (IL)-1β or tumor necrosis factor-α (TNF-α) (positive controls; 10 ng/mL), irisin (5, 10, 25 ng/mL), IL-1β or TNF-α pretreatment and subsequent co-treatment with irisin, pretreatment with irisin and subsequent co-treatment with IL-1β or TNF-α. hTC metabolic activity, proliferation, and nitrite production were evaluated. Detection of unphosphorylated and phosphorylated p38 and ERK was performed. Tissue samples were analyzed by histology and immunohistochemistry to evaluate irisin αVβ5 receptor expression. Irisin significantly increased hTC proliferation and metabolic activity, while reducing the production of nitrites both before and after the addition of IL-1β and TNF-α. Interestingly, irisin reduced p-p38 and pERK levels in inflamed hTCs. The αVβ5 receptor was uniformly expressed on hTC plasma membranes, supporting the potential binding of irisin. This is the first study reporting the capacity of irisin to target hTCs and modulating their response to inflammatory stresses, possibly orchestrating a biological crosstalk between the muscle and tendon., (© 2023 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Long-term chronic inflammation after Achilles tendon injury is critical for tendinopathy. Platelet-rich plasma (PRP) injection, which is a common method for treating tendinopathy, has positive effects on tendon repair. In addition, tendon-derived stem cells (TDSCs), which are stem cells located in tendons, play a major role in maintaining tissue homeostasis and postinjury repair. In this study, injectable gelatine methacryloyl (GelMA) microparticles containing PRP laden with TDSCs (PRP-TDSC-GM) were prepared by a projection-based 3D bioprinting technique. Our results showed that PRP-TDSC-GM could promote tendon differentiation in TDSCs and reduce the inflammatory response by downregulating the PI3K-AKT pathway, thus promoting the structural and functional repair of tendons in vivo., (© 2023. The Author(s).)

Tendinopathy is a disease with surging prevalence. Lacking understanding of molecular mechanisms impedes the development of therapeutic approaches and agents. Lysine lactylation (Kla) is a newly discovered post-translational modification related to glycolysis. It has long been noted that manipulation of glycolysis metabolism could affect tendon cell function, tendon homeostasis, and healing process of tendon. However, protein lactylation sites in tendinopathy remain unexplored. Here, we conducted the first proteome-wide Kla analysis in tendon samples harvested from patients with rotator cuff tendinopathy (RCT), which identified 872 Kla sites across 284 proteins. Compared with normal counterparts, 136 Kla sites on 77 proteins were identified as upregulated in the pathological tendon, while 56 sites on 32 proteins were downregulated. Function enrichment analysis demonstrated that the majority of proteins with upregulated Kla levels functioned in organization of the tendon matrix and cholesterol metabolism, accompanied by lower expression levels which meant impaired cholesterol metabolism and degeneration of the tendon matrix, indicating potential cross-talk between protein lactylation and expression levels. At last, by western blotting and immunofluorescence, we verified the correlation between high lactylation and the downregulation of matrix and cholesterol-related proteins including BGN, MYL3, TPM3, and APOC3. ProteomeXchange: PXD033146.

Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1β, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells' ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1β using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1β stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB., (© 2022. The Author(s).)

The development and progression of rotator cuff tendinopathy (RCT) is multifactorial and likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in RCT, including the role of histone modification, is not well established. Using chromatin immunoprecipitation sequencing, differences in the trimethylation status of H3K4 and H3K27 histones in late-stage RCT compared to control were investigated in this study. For H3K4, 24 genomic loci were found to be significantly more trimethylated in RCT compared to control ( p < 0.05), implicating genes such as DKK2 , JAG2 , and SMOC2 in RCT. For H3K27, 31 loci were shown to be more trimethylated ( p < 0.05) in RCT compared to control, inferring a role for EPHA3 , ROCK1 , and DEFβ115 . Furthermore, 14 loci were significantly less trimethylated ( p < 0.05) in control compared to RCT, implicating EFNA5 , GDF6 , and GDF7 . Finally, the TGFβ signaling, axon guidance, and regulation of focal adhesion assembly pathways were found to be enriched in RCT. These findings suggest that the development and progression of RCT is, at least in part, under epigenetic control, highlighting the influence of histone modifications in this disorder and paving the way to further understand the role of epigenome in RCT.

The erroneous differentiation of tendon-derived stem cells (TDSCs) into adipocytes, chondrocytes, and osteoblasts is believed to play an important role in the development of tendinopathy. However, the regulatory mechanisms of TDSC differentiation remain unclear. The aim of this study is to investigate the contribution and mechanism of the tendon microstructural disruption to the differentiation of TDSCs. Bovine Achilles tendons were sliced. The tendon slices were stretched with different tensile strains to mimic the tendon structure alteration at various scales. The TDSCs were cultured on the tendon slices. The differentiation of TDSCs and endoplasmic reticulum (ER) stress in the TDSCs were investigated with quantitative reverse transcription polymerase chain reaction, immunostaining and western blot. The effect of ER stress inhibition on chondrogenic differentiation of the TDSCs was further investigated. The structural alteration did not affect the viability of TDSCs. However, the structural alteration of tendon slices with 6.4% strain promoted TDSCs to express the chondrogenic genes. ER stress-related markers, ATF-4 and PERK, were also upregulated. With the inhibition of ER stress, the expression of ATF-4 and the chondrogenic gene SOX9 of TDSCs were inhibited. The study indicated that tendon microdamage could induce the chondrogenic differentiation of TDSCs through triggering ER stress to activate ATF-4 and SOX9 subsequently., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

This study aimed to evaluate the effectiveness of omega-3 supplementation with exercise in a collagenase-induced Achilles tendinopathy (AT) rat model. Experimental groups (healthy control (HC), AT, exercise (Ex), omega-3 (W), and Ex+W) were randomly allocated. After a week of adaptation, oral omega-3 was initiated for 8 weeks (5 days/week). The exercise groups performed treadmill running for 30 min/day (5 days/week, 20 m/min, 8 weeks) following one week of adaptation (10 m/min, 15 min/day). Matrix metalloproteinase-13 (MMP-13), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), and total antioxidant-oxidant status (TAS) levels were determined in serum samples. Tendon samples were obtained for biomechanical, histopathological, and immunohistochemical assessments. Ultimate tensile force, yield force, stiffness values, collagen type-I alpha 1 expression, and serum TAS significantly decreased ( P  < 0.05) in AT vs. HC. These values and expression significantly increased in the Ex+W group vs. AT. Serum MMP-13, IL-1β, and TNF-α levels decreased in all treatment groups vs. AT. The most significant decrease was found in the Ex+W group ( P  < 0.01). Histopathologically, the improvement in degeneration was statistically significant in the Ex+W group ( P  < 0.05). Immunohistochemically, MMP-13, IL-1β, TNF-α, and nitric oxide synthase-2 expression was decreased in all treatment groups vs. AT. In conclusion, omega-3 and exercise might be recommended in AT patients.

It has been reported that tendon-derived stem cells(TDSCs) conduce to the ostosis in tendon diseases, and the molecular mechanism needs to be discussed. To investigate the function and mechanism of LncRNA in tendinopathy. Tendon of tendinopathy patients and health controls were obtained, and sequencing analysis have been performed to detect the significantly expressed genes and non-coding RNAs. Moreover, to further discuss LncRNA AC108925 in tendinopathy, tendinopathy animal models have been established, and the expression of LncRNA AC108925 expression was examined by RT-qPCR methods. Furthermore, hTDSCs have been treated by osteogenic medium, and the modulating function of LncRNA AC108925 on the osteoblast differentiation of hTDSCs have been examined. Sequencing analysis showed that AC108925 a dramatically elevated LncRNA, and results of animal and cells studies confirmed the finding. Knockdown AC108925 inhibited the osteogenic differentiation of osteogenic medium treated TDSCs by decreasing the expression of osteogenic markers. Furthermore, miR-146a-3p is a target of AC108925 in TDSCs, and miR-146a-3p is a negative modulator of osteogenic differentiation of hTDSCs by inhibiting the effects of AC108925 shRNA on osteogenic differentiation of hTDSCs. AC108925 can regulate the osteogenic differentiation of hTDSCs via regulating the miR-146a-3p. Targeting the AC108925/miR-146a-3p axis might be a latent way to treat tendinopathy., Competing Interests: Declaration of Competing Interest The authors declare there is no conflict of interests., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Actin is a central mediator between mechanical force and cellular phenotype. In tendons, it is speculated that mechanical stress deprivation regulates gene expression by reducing filamentous (F)-actin. However, the mechanisms regulating tenocyte F-actin remain unclear. Tropomyosins (Tpms) are master regulators of F-actin. There are more than 40 Tpm isoforms, each having the unique capability to stabilize F-actin subpopulations. We investigated F-actin polymerization in stress-deprived tendons and tested the hypothesis that stress fiber-associated Tpm(s) stabilize F-actin to regulate cellular phenotype. Stress deprivation of mouse tail tendon down-regulated tenogenic and up-regulated protease (matrix metalloproteinase-3) mRNA levels. Concomitant with mRNA modulation were increases in G/F-actin, confirming reduced F-actin by tendon stress deprivation. To investigate the molecular regulation of F-actin, we identified that tail, Achilles, and plantaris tendons express three isoforms in common: Tpm1.6, 3.1, and 4.2. Tpm3.1 associates with F-actin in native and primary tenocytes. Tpm3.1 inhibition reduces F-actin, leading to decreases in tenogenic expression, increases in chondrogenic expression, and enhancement of protease expression in mouse and human tenocytes. These expression changes by Tpm3.1 inhibition are consistent with tendinosis progression. A further understanding of F-actin regulation in musculoskeletal cells could lead to new therapeutic interventions to prevent alterations in cellular phenotype during disease progression.

Tendinopathy is a common disorder that leads to pain and impaired quality of life. Recent studies revealed that osteogenic differentiation of tendon stem/progenitor cells (TSPCs) played an important role in the pathogenesis of tendon calcification and tendinopathy. In this study, we found that the growth hormone-releasing hormone agonist (GA) can prevent matrix degradation and osteogenic differentiation in TSPCs. As oxidative stress is a key factor in the osteogenic differentiation of TSPCs, we used bovine serum albumin/heparin nanoparticles (BHNPs), which have biocompatibility and drug loading capacity, to scavenge reactive oxygen species (ROS) and achieve sustained release of GA at the site of inflammation. The newly developed BHNPs@GA had a synergetic effect on reducing ROS production in TSPCs. In addition, BHNPs@GA effectively inhibited tendon calcification and promoted collagen formation in a rat model of tendinopathy. Focusing on the ROS underlying the differentiation and dedifferentiation of TSPCs, this work demonstrated that sustained release of GA targeting ROS and ectopic ossification is a practical therapeutic strategy for treating tendinopathy. STATEMENT OF SIGNIFICANCE: Osteogenic differentiation of tendon stem/progenitor cells (TSPCs) plays an important role in the pathogenesis of ectopic calcification in tendinopathy. In this study, we found that growth hormone-releasing hormone agonist (GA) can reduce reactive oxygen species (ROS) production and adjust TSPCs differentiation. Bovine serum albumin/heparin nanoparticles (BHNPs) were developed to encapsulate GA and achieve sustained release of GA at the site of inflammation. The developed compound, BHNPs@GA, with a synergistic effect of inhibiting ROS and thus, can effectively adjust TSPCs differentiation, inhibit tendon calcification, and promote collagen formation in tendinopathy. This study highlighted the role of ROS underlying the differentiation and dedifferentiation of TSPCs in tendinopathy, and findings may help to identify new therapeutic targets and develop novel strategy for treating tendinopathy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that may influence the paper., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Objective: To establish and validate an intact rotator cuff rat model for exploring the pathophysiological effects of type 2 diabetes on the rotator cuff tendon in vivo ., Methods: A total of 45 adult male rats were randomly divided into a control group (n = 9) and type 2 diabetes group (n=36). The rats were sacrificed at 2 weeks (T2DM-2w group, n=9), 4 weeks (T2DM-4w group, n=9), 8 weeks (T2DM-8w group, n=9), and 12 weeks (T2DM-12w group, n=9) after successful modeling of type 2 diabetes. Bilateral shoulder samples were collected for gross observation and measurement, protein expression(enzyme-linked immunosorbent assay,ELISA), histological evaluation, biomechanical testing, and gene expression (real-time quantitative polymerase chain reaction, qRT-PCR)., Results: Protein expression showed that the expression of IL-6 and Advanced glycation end products (AGEs)in serum increased in type 2 diabetic group compared with the non-diabetic group. Histologically, collagen fibers in rotator cuff tendons of type 2 diabetic rats were disorganized, ruptured, and with scar hyperplasia, neovascularization, and extracellular matrix disturbances, while Bonar score showed significant and continuously aggravated tendinopathy over 12 weeks. The biomechanical evaluation showed that the ultimate load of rotator cuff tendons in type 2 diabetic rats gradually decreased, and the ultimate load was negatively correlated with AGEs content. Gene expression analysis showed increased expression of genes associated with matrix remodeling (COL-1A1), tendon development (TNC), and fatty infiltration (FABP4) in tendon specimens from the type 2 diabetic group., Conclusion: Persistent type 2 diabetes is associated with the rupture of collagen fiber structure, disturbance in the extracellular matrix, and biomechanical decline of the rotator cuff tendon. The establishment of this new rat model of rotator cuff tendinopathy provides a valuable research basis for studying the cellular and molecular mechanisms of diabetes-induced rotator cuff tendinopathy., 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 © 2022 Xu, Zhang, Ren, Wang, Zhang, Zhao and Yu.)

Tendinopathy is a common tendon disorder characterized by pain, swelling, and dysfunction. Current evidence has demonstrated that the depletion of stem cell pool and non-tenogenic differentiation of tendon-derived stem/progenitor cells (TSPCs) might account for the pathogenesis of tendinopathy. FNDC5/Irisin, as a novel exercise-induced myokine, is proved to be involved in the exercise-induced protective effects on musculoskeletal disorders. However, whether irisin can affect TSPCs fate is still unknown. To ascertain the roles of irisin on the proliferation and tenogenic differentiation of TSPCs, rat TSPCs were isolated and incubated with irisin. Cell viability, phenotypic changes, and related signaling pathways were evaluated by CCK-8 assay, colony formation assay, real-time PCR, Western blot, immunofluorescence, and proteasome activity assay. We found that irisin treatment increased the proliferative and colony-forming abilities, and promoted the tenogenic differentiation of TSPCs by upregulating the expression of YAP/TAZ. In conclusion, our work showed for the first time that irisin promotes the proliferation and tenogenic differentiation of rat TSPCs in vitro by activating YAP/TAZ, and the process was associated with a ubiquitin-proteasome proteolytic pathway. In conclusion, irisin and agents targeting YAP/TAZ may be promising therapeutic options for tendinopathy., (© 2022. The Author(s).)

Rotator cuff tendinopathy (RCT) is the primary reason for shoulder surgery and its clinical management is still challenging. Hyaluronic acid (HA) has been shown to have anti-inflammatory effects in vitro and in vivo under RCT conditions, characterized by an exaggerated oxidative stress (OS). However, molecular mechanisms underlying HA-related effects are still partially disclosed. With these aims, a cell model of RCT was established by exposing primary human tenocytes to H 2 O 2 for up to 72 h. Four different HAs by molecular weight were administered to measure nitric oxide (NO) and OS, apoptosis, and collagen 1 expression. In parallel, the well-known antioxidant ascorbic acid was administered for comparison. The present study highlights that HAs characterized by a low molecular weight are able to counteract the H 2 O 2 -induced OS by decreasing the percentage of apoptotic cells and reversing the activation of caspase 3 and 7. Likewise, NO intracellular levels are comparable to the ones of controls. In parallel, collagen 1 expression was ameliorated by HAs characterized by higher molecular weights compared to AA. These findings confirm that HA plays an antioxidant role comparable to AA depending on the molecular weight, and highlight the molecular mechanisms underlying the HA anti-apoptotic effects.

Background: Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have gained momentum as a treatment for tendinopathy. Multiple studies have demonstrated significant differences in cargo composition between the 2 subtypes of MSC-EVs (ie, exosomes and ectosomes), which may result in different therapeutic effects. However, the effects of the 2 EV subtypes on tendinopathy have not yet been compared., Purpose: To compare the effects of adipose stem cell-derived exosomes (ASC-Exos) and ectosomes (ASC-Ectos) on Achilles tendinopathy., Study Design: Controlled laboratory study., Methods: Rats were administered collagenase injections to generate a model of Achilles tendinopathy. A week later, 36 rats were randomly assigned to 3 groups. In each group, Achilles tendons were injected with equal volumes of ASC-Exos, ASC-Ectos, or saline (12 legs/group). The healing outcomes were evaluated by magnetic resonance imaging, histology, immunohistochemistry, transmission electron microscopy, and biomechanical testing at 3 and 5 weeks after collagenase injection., Results: At 3 and 5 weeks, the ASC-Exo group had better histological scores ( P = .0036 and P = .0276, respectively), a lower fibril density ( P < .0001 and P = .0310, respectively), and a larger collagen diameter ( P = .0052 and P < .0001, respectively) than the ASC-Ecto group. At 5 weeks, the expression of collagen type 1 and CD206 in the ASC-Exo group was significantly higher than that in the ASC-Ecto group ( P = .0025 and P = .0010, respectively). Regarding biomechanical testing, the ASC-Exo group showed higher failure load ( P = .0005), tensile stress ( P < .0001), and elastic modulus ( P < .0001) than the ASC-Ecto group., Conclusion: ASC-Exos had more beneficial effects on tendon repair than ASC-Ectos in a rat model of Achilles tendinopathy., Clinical Relevance: Administration of ASC-EVs may have the potential to treat Achilles tendinopathy, and delivery of ASC-Exos could provide additional benefits. It is necessary to compare the healing responses caused by different EV subtypes to further understand their effects on tendinopathy and to aid clinical decision making.

The pathophysiology of pain in patients suffering from rotator cuff (RC) tendinopathy or tears has been examined in various ways. Several molecules from tissue samples taken from the subacromial bursa, supraspinatus tendon, glenohumeral joint fluid, and synovium as well as from peripheral blood have been investigated. This article explores these studies, the assessed biomarkers, and groups their results according to the status of tendon integrity (tendinopathy or tear). Through a structured PubMed database search, 9 out of 658 articles were reviewed. Interleukins, mostly IL-1b and its antagonist, IL-1ra, matrix Metalloproteinases (MMPs), the vascular endothelial growth factor (VEGF) and TNF-a are biomarkers directly searched for correlation to pain level. Most studies agree that IL-1b is directly positively correlated to the degree of pain in patients with RC tendinopathy, especially when the examined sample is taken from the subacromial bursa. VEGF, and TNF-a have been related to shoulder pain preoperatively and TNF-a has also been linked with sleep disturbance. Further studies pointing to more biomarkers taken from the subacromial bursa or tendon directly relating to pain degree are warranted.

Background: Degenerative rotator cuff tendinopathy (RCT) is associated with the senescence of tendon-derived stem cells (TDSCs). Nonsteroidal anti-inflammatory drugs have been demonstrated to alleviate age-associated inflammation (inflamm-aging)-induced cellular senescence of skeletal stem/progenitor cells. However, whether they can alleviate degenerative RCT through reducing inflamm-aging-related TDSC senescence is still unknown., Purpose: To assess whether celecoxib can prevent the inflamm-aging-related cellular senescence of TDSCs., Study Design: Controlled laboratory study., Methods: TDSCs were isolated from degenerative RCT tendons (S-TDSCs) and healthy hamstring tendons (Y-TDSCs), and the cellular senescence of TDSCs was evaluated. Thereafter, the senescent TDSC-conditioned medium (SEN-CM) was collected to culture Y-TDSCs with or without celecoxib. The effects of celecoxib on TDSC senescence were examined by assaying the expression of aging-related markers. Furthermore, the level of the NF-κB pathway was determined by Western blot analysis to explore the underlying mechanism. Its effects on preventing dysfunction of inflamm-aging-induced senescent TDSCs were also determined using multilineage differentiation assay., Results: S-TDSCs showed increased senescence-associated β-galactosidase activity and enhanced expression of γ-H2AX , p21 CIP1A , p16 INK4A , and senescence-associated secretory phenotype factors. SEN-CM accelerated the senescence progress of Y-TDSCs, resulting in an increase in senescence markers. To some extent, celecoxib treatment could prevent the detrimental effects of inflamm-aging on Y-TDSCs. The level of the NF-κB pathway was increased in the SEN-CM group but decreased with the use of celecoxib. Moreover, the reduced senescence of TDSCs resulted in preservation of the TDSC tenogenic potential., Conclusion: Celecoxib treatment can prevent inflamm-aging-induced TDSC senescence, which holds potential for alleviating the development of degenerative RCT., Clinical Relevance: In addition to relieving the symptoms of patients with RCT, treatment with celecoxib, a common nonsteroidal anti-inflammatory drug, may defer the development of RCT and prevent rotator cuff tears by delaying TDSC senescence.

Tendinopathy is mainly characterized by local pain, functional limitation and decreased athletic ability, which seriously affects the quality of life of patients and the career of athletes. Farrerol (FA), one of the main active compounds extracted from Rhododendron and plants in the Rhododendron family, has a wide range of pharmacological activities, such as immunomodulatory, anti-inflammatory and antiviral effects. However, the effect of FA on tendinopathy is unclear. Here, we investigated the pharmacological effect and mechanism of FA in tendon injury through collagenase-induced tendinopathy in vivo and RSL3-induced tenocytes injury in vitro. The results showed that FA alleviated the infiltration of inflammatory cells, promoted tenogenesis and improved mechanical properties of the Achilles tendon in rats. In addition, ferroptosis inducer RSL3 inhibits the tenogenesis in vitro and in vivo, which accelerates the progression of tendinopathy. Moreover, FA effectively inhibited iron accumulation and alleviated ferroptosis in the Achilles tendon. Using in vitro experiments, we found that FA antagonized ferroptosis by reducing lipid peroxidation and iron accumulation in tenocytes. Finally, we found that glutathione peroxidase 4 silencing could block the protective effect of FA on ferroptosis of tenocytes. Therefore, the results of this study suggest that FA can relieve collagenase-induced tendinopathy by inhibiting ferroptosis, and reveal that FA may be a potentially effective drug for the treatment of tendinopathy in the future., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The effect of mechanical load on tendinopathic tissue is usually studied in the context of identifying mechanisms responsible for tendon degradation. However, loading is also one of the most common treatments for tendinopathy. It is therefore possible that different loads result in different cellular responses within a tendon. To test this hypothesis, we first established a rodent model of tendinopathy that has a transcriptional signature similar to human tendinopathy. Tendinopathy was modeled in the rat by producing a lesion in the central core of the patellar tendon using a biopsy punch, followed by two weeks to allow scar formation. We performed 3' Tag RNA-Seq to identify genes that were differentially expressed between the native and scarred rat patellar tendon. Genes involved in extracellular matrix (ECM) structure and turnover were increased, mitochondrial genes were decreased, and there was no inflammatory signature in the tendinopathic tissue. These transcriptional changes phenocopy previously published whole transcriptome analysis in human tendinopathy. After validating the model, the initial response to injury and loading was determined. Two weeks after creation of the patellar tendon lesion, the tendon was loaded using either 4 × 30s isometric or a time-under-tension matched (360 × 0.33s) dynamic protocol. Injured +/- loading and contralateral control tendons were collected eighteen hours after loading, RNA was extracted, and gene expression was quantified using qRT-PCR of the scar with or without loading. The expression of scleraxis and type I collagen increased following isometric loading relative to those loaded dynamically. By contrast, the expression of type II collagen increased in the dynamic samples relative to those loaded isometrically. These data suggest that dynamic loading of a central core tendon injury increases fibrocartilage markers, whereas long isometric loads stimulate markers of tendon regeneration., Competing Interests: Declaration of competing interests KB has received an honorarium, support for travel, and a research contract from PepsiCo on a nutritional intervention tangentially related to the current work. KB is paid by professional sports and Olympic teams to consult on tendon issues. DS and MM have no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Tendons are vital collagen-dense specialized connective tissues transducing the force from skeletal muscle to the bone, thus enabling movement of the human body. Tendon cells adjust matrix turnover in response to physiological tissue loading and pathological overloading (tendinopathy). Nevertheless, the regulation of tendon matrix quality control is still poorly understood and the pathogenesis of tendinopathy is presently unsolved. Autophagy, the major mechanism of degradation and recycling of cellular components, plays a fundamental role in the homeostasis of several tissues. Here, we investigate the contribution of autophagy to human tendons' physiology, and we provide in vivo evidence that it is an active process in human tendon tissue. We show that selective autophagy of the endoplasmic reticulum (ER-phagy), regulates the secretion of type I procollagen (PC1), the major component of tendon extracellular matrix. Pharmacological activation of autophagy by inhibition of mTOR pathway alters the ultrastructural morphology of three-dimensional tissue-engineered tendons, shifting collagen fibrils size distribution. Moreover, autophagy induction negatively affects the biomechanical properties of the tissue-engineered tendons, causing a reduction in mechanical strength under tensile force. Overall, our results provide the first evidence that autophagy regulates tendon homeostasis by controlling PC1 quality control, thus potentially playing a role in the development of injured tendons., (© 2022. The Author(s).)

The term tendinopathy indicates a wide spectrum of conditions characterized by alterations in tendon tissue homeostatic response and damage to the extracellular matrix. The current pharmacological approach involves the use of nonsteroidal anti-inflammatory drugs and corticosteroids often with unsatisfactory results, making essential the identification of new treatments. In this study, the pro-regenerative and protective effects of an aqueous fibroin solution (0.5-500 μg/mL) against glucose oxidase (GOx)-induced damage in rat tenocytes were investigated. Then, fibroin anti-hyperalgesic and protective actions were evaluated in two models of tendinopathy induced in rats by collagenase or carrageenan injection, respectively. In vitro, 5-10 μg/mL fibroin per se increased cell viability and reverted the morphological alterations caused by GOx (0.1 U/mL). Fibroin 10 μg/mL evoked proliferative signaling upregulating the expression of decorin, scleraxin, tenomodulin (p < 0.001), FGF-2, and tenascin-C (p < 0.01) genes. Fibroin enhanced the basal FGF-2 and MMP-9 protein concentrations and prevented their GOx-mediated decrease. Furthermore, fibroin positively modulated the production of collagen type I. In vivo, the peri-tendinous injection of fibroin (5 mg) reduced the development of spontaneous pain and hypersensitivity (p < 0.01) induced by the intra-tendinous injection of collagenase; the efficacy was comparable to that of triamcinolone. The pain-relieving action of fibroin (peri-tendinous) was confirmed in the model of tendinopathy induced by carrageenan (intra-tendinous) where this fibrous protein was also able to improve tendon matrix organization, normalizing the orientation of collagen fibers. In conclusion, the use of fibroin in tendinopathies is suggested taking advantage of its excellent mechanical properties, pain-relieving effects, and ability to promote tissue regeneration processes., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aim: This study aimed to explore the effect of small extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells (iMSC-sEVs) on acute pain and investigate the underlying mechanisms. Materials & methods: The pathology of tendons was accessed by hematoxylin and eosin staining, immunohistochemical and immunofluorescent staining. The pain degree was measured by pain-related behaviors. In vitro , we performed β-hexosaminidase release assay, RT-qPCR, toluidine blue staining, ELISA and RNA sequencing. Results: iMSC-sEVs effectively alleviated acute pain in tendinopathy as well as inhibiting activated mast cell infiltration and interactions with nerve fibers in vivo . In vitro , iMSC-sEVs reduced the degranulation of mast cells and the expression of proinflammatory cytokines and genes involved in the HIF-1 signaling pathway. Conclusion: This study demonstrated that iMSC-sEVs relieved tendinopathy-related pain through inhibiting mast cell activation via the HIF-1 signaling pathway.

Tendon injuries are among the most common ailments of the musculoskeletal system. Prolonged inflammation and persistent vasculature are common complications associated with poor healing. Damaged tendon, replaced with scar tissue, never completely regains the native structural or biomechanical properties. This study evaluated the effects of micronized dehydrated human amnion/chorion membrane (μdHACM) on the inflammatory environment and hypervascularity associated with tendinopathy. Stimulation of human tenocytes with interleukin-1 beta (IL1β) induced the expression of inflammatory and catabolic markers, resulting in secretion of active MMPs and type 3 collagen that is associated with a degenerative phenotype. Treatment with μdHACM diminished the effects of IL1β, reducing the expression of inflammatory genes, proteases, and extracellular matrix components, and decreasing the presence of active MMP and type 3 collagen. Additionally, a co-culture model was developed to evaluate the effects of μdHACM on angiogenesis associated with tendinopathy. Micronized dHACM differentially regulated angiogenesis depending upon the cellular environment in which it was placed. This phenomenon can be explained in part through the detection of both angiogenic protagonists and antagonists in μdHACM. Observations from this study identify a mechanism by which μdHACM regulates inflammatory processes and angiogenesis in vitro, two key pathways implicated in tendinopathic injuries., (© 2021 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals LLC.)

Calebin A (CA) is one of the active constituents of turmeric and has anti-inflammatory and antioxidant effects. Excessive inflammation and cell apoptosis are the main causes of tendinitis and tendinopathies. However, the role of CA in tendinitis is still unclear and needs to be studied in detail. Tenocytes in monolayer or 3D-alginate cultures in the multicellular tendinitis microenvironment (fibroblast cells) with T-lymphocytes (TN-ME) or with TNF-α or TNF-β, were kept without treatment or treated with CA to study their range of actions in inflammation. We determined that CA blocked TNF-β-, similar to TNF-α-induced adhesiveness of T-lymphocytes to tenocytes. Moreover, immunofluorescence and immunoblotting showed that CA, similar to BMS-345541 (specific IKK-inhibitor), suppressed T-lymphocytes, or the TNF-α- or TNF-β-induced down-regulation of Collagen I, Tenomodulin, tenocyte-specific transcription factor (Scleraxis) and the up-regulation of NF-κB phosphorylation; thus, its translocation to the nucleus as well as various NF-κB-regulated proteins was implicated in inflammatory and degradative processes. Furthermore, CA significantly suppressed T-lymphocyte-induced signaling, similar to TNF-β-induced signaling, and NF-κB activation by inhibiting the phosphorylation and degradation of IκBα (an NF-κB inhibitor) and IκB-kinase activity. Finally, inflammatory TN-ME induced the functional linkage between NF-κB and Scleraxis, proposing that a synergistic interaction between the two transcription factors is required for the initiation of tendinitis, whereas CA strongly attenuated this linkage and subsequent inflammation. For the first time, we suggest that CA modulates TN-ME-promoted inflammation in tenocytes, at least in part, via NF-κB/Scleraxis signaling. Thus, CA seems to be a potential bioactive compound for the prevention and treatment of tendinitis.

Background: Achilles tendinopathy, seen in athletes and manual labor workers, is an inflammatory condition characterized by chronic tendon pain. Owing to the toxicity that develops in various organs attributed to the use of anti-inflammatory drugs, there is a need for new therapeutic agents., Purpose: In the present study, the effects of quercetin (Que), the one that attracted the most attention of researchers studying this group of flavonoids, were investigated against collagenase-induced tendinopathy., Study Design: Controlled laboratory study., Methods: A total of 35 Sprague-Dawley rats were used in the study. Tendinopathy was created by injecting a single dose of collagenase (10 μL; 10 mg/mL) into the tendons of rats. Thirty minutes after the injection, Que was administered at doses of 25 or 50 mg/kg. Que administration was carried out for 7 days. Animals underwent a motility test at the end of the study. In addition, markers of oxidative stress, inflammation, apoptosis, and autophagy, as well as the expression levels of matrix metalloproteinases (MMPs 2, 3, 9, and 13), ICAM-1, and STAT3, were measured in tendon tissues with biochemical, molecular, and Western blot techniques., Results: The results showed that oxidative stress, inflammation, apoptosis, and autophagy were triggered by the injection of collagenase. In addition, MMPs, ICAM-1, and STAT3 were activated to participate in the development of tendinopathy. Que was found to reduce ICAM-1 levels in tendon tissue. Moreover, Que showed antioxidant, anti-inflammatory, antiapoptotic, and antiautophagic effects on tendons against tendinopathy. More important, Que suppressed the expression of MMPs in the tendon tissues., Conclusion: Que has protective properties against collagenase-induced tendon damage in rats., Clinical Relevance: We believe that with further study, Que may be shown to be an alternative treatment option for athletes or others who experience tendon injuries.