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Postoperative tissue adhesion and poor tendon healing are major clinical problems associated with tendon surgery. To avoid postoperative adhesion and promote tendon healing, we developed and synthesized a membrane to wrap the surgical site after tendon suturing. The bilayer-structured porous membrane comprised an outer layer [1,4-butanediol diglycidyl ether cross-linked with carboxymethyl cellulose (CX)] and an inner layer [1,4-butanediol diglycidyl ether cross-linked with Bletilla striata polysaccharides and carboxymethyl cellulose (CXB)]. The morphology, chemical functional groups, and membrane structure were determined. In vitro experiments revealed that the CX/CXB membrane demonstrated good biosafety and biodegradability, promoted tenocyte proliferation and migration, and exhibited low cell attachment and anti-inflammatory effects. Furthermore, in in vivo animal study, the CX/CXB membrane effectively reduced postoperative tendon-peripheral tissue adhesion and improved tendon repair, downregulating inflammatory cytokines in the tendon tissue at the surgical site, which ultimately increased tendon strength by 54% after 4 weeks.

G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout (G9a cKO) mice. We crossed Sox9 Cre/+ mice with G9a fl/fl mice to generate G9a fl/fl ; Sox9 Cre/+ mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin (Tnmd), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development., (© 2024. The Author(s).)

Allogeneic serum and tissue-specific extracellular matrix have been shown to maintain permanently differentiated cell phenotype in culture. This is of particular importance for human tenocytes, a cell population that readily loses its function during ex vivo culture. With these in mind, herein we extracted human tenocytes using either foetal bovine serum or human serum, cultured them in the absence and presence of carrageenan and Ficoll®, the most widely used macromolecular crowding agents (to induce tissue-specific extracellular matrix deposition), and assessed cellular function, via metabolic activity, viability, proliferation and immunofluorescence for collagen related molecules, non-collagenous molecules and transmembrane molecules. At day 7, longest time point assessed, neither carrageenan nor Ficoll® significantly affected metabolic activity, viability and proliferation in either serum and human serum significantly increased metabolic activity and proliferation. At day 7, in the absence of macromolecular crowding, cells in human serum deposited significantly lower collagen type VI, biglycan, versican and tenomodulin than cells in foetal bovine serum. Interestingly, at day 7, in comparison to the no macromolecular crowding group, carrageenan in foetal bovine serum induced the highest effect, as judged by the highest number of significantly increased molecules (collagen type I, collagen type IV, collagen type V, collagen type VI, transforming growth factor β1, matrix metalloproteinase 14, lumican, versican, scleraxis and integrin α2β1). These data, although contradict previous observations where human serum outperformed foetal bovine serum, at the same time, support the use of foetal bovine serum in the development of cell-based medicines., 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 Authors. Published by Elsevier GmbH.. All rights reserved.)

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 leading cause of mobility issues. Currently, the cell-matrix interactions involved in the development of tendinopathy are not fully understood. In vitro tendon models provide a unique tool for addressing this knowledge gap as they permit fine control over biochemical, micromechanical, and structural aspects of the local environment to explore cell-matrix interactions. In this study, direct-write, near-field electrospinning of gelatin solution was implemented to fabricate micron-scale fibrous scaffolds that mimic native collagen fiber size and orientation. The stiffness of these fibrous scaffolds was found to be controllable between 1 MPa and 8 MPa using different crosslinking methods (EDC, DHT, DHT+EDC) or through altering the duration of crosslinking with EDC (1 h to 24 h). EDC crosslinking provided the greatest fiber stability, surviving up to 3 weeks in vitro. Differences in stiffness resulted in phenotypic changes for equine tenocytes with low stiffness fibers (∼1 MPa) promoting an elongated nuclear aspect ratio while those on high stiffness fibers (∼8 MPa) were rounded. High stiffness fibers resulted in the upregulation of matrix metalloproteinase (MMPs) and proteoglycans (possible indicators for tendinopathy) relative to low stiffness fibers. These results demonstrate the feasibility of direct-written gelatin scaffolds as tendon in vitro models and provide evidence that matrix mechanical properties may be crucial factors in cell-matrix interactions during tendinopathy formation., (Copyright © 2024 by ASME.)

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 injuries typically display limited reparative capacity, often resulting in suboptimal outcomes and an elevated risk of recurrence or rupture. While cytokines of the IL-6 family are primarily recognised for their inflammatory properties, they also have multifaceted roles in tissue regeneration and repair. Despite this, studies examining the association between IL-6 family cytokines and tendon repair remained scarce. gp130, a type of glycoprotein, functions as a co-receptor for all cytokines in the IL-6 family. Its role is to assist in the transmission of signals following the binding of ligands to receptors. RCGD423 is a gp130 modulator. Phosphorylation of residue Y759 of gp130 recruits SHP2 and SOCS3 and inhibits activation of the STAT3 pathway. In our study, RCGD423 stimulated the formation of homologous dimers of gp130 and the phosphorylation of Y759 residues without the involvement of IL-6 and IL-6R. Subsequently, the phosphorylated residues recruited SHP2, activating the downstream ERK and AKT pathways. These mechanisms ultimately promoted the migration ability of tenocytes and matrix synthesis, especially collagen I. Moreover, RCGD423 also demonstrated significant improvements in collagen content, alignment of collagen fibres, and biological and biomechanical function in a rat Achilles tendon injury model. In summary, we demonstrated a promising gp130 modulator (RCGD423) that could potentially enhance tendon injury repair by redirecting downstream signalling of IL-6, suggesting its potential therapeutic application for tendon injuries., 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 Elsevier Inc. All rights reserved.)

Objectives: Autologous tenocyte implantation (OrthoATI™) therapy has demonstrated efficacy in treating patients with tendinopathy at various anatomical sites. This study evaluates the effect of patient age, gender, and tendon biopsy site on morphology, growth, and gene expression of autologous tendon cells used to treat chronic tendinopathy., Methods: Patients undergoing OrthoATI™ for tendinopathies between 2020 and 2022 were initially treated by biopsies taken from patella tendon (PT) or palmaris longus tendon (PL). The biopsies were sent to a Good Manufacturing Practice (GMP) cell laboratory where tendon cells were isolated, cultured, and expanded for four to six weeks. Cell morphology was assessed using phase contrast microscopy. Droplet digital PCR (ddPCR) was utilized for gene expression analysis. Dichotomous results were compared between groups using x 2 or Fisher's exact tests with no adjustment for multiple comparisons. The nonparametric Mann-Whitney U and Kruskal-Wallis tests were utilized for the sex and age (<35y, 35-44y, 45-54y, >55y) analyses, respectively. All analyses were performed using IBM SPSS v27, and a two-tailed P-value of <0.05 was considered statistically significant., Results: 149 patients were included in the analysis. The PT was biopsied in 63 patients, and PL in 86 patients. There were no observer effects for age and gender between the PT and PL groups. There was no statistical significance between the PT and PL tendons for cell morphology, average cell population doubling time (PDT) (PT 83.9 vs PL 82.7 ​h, p ​= ​0.482), cellular yield (PT 16.2 vs PL 15.2 ​× ​10 6 , p ​= ​0.099), and cell viability (PT 98.7 vs PL 99.0%, p ​= ​0.277). Additionally, ddPCR analyses showed no statistical significance found in tenogenic gene expression, including collagen type I (COL1, p ​= ​0.86), tenomodulin (TNMD, p ​= ​0.837) and scleraxis (SCX, p ​= ​0.331) between PT- and PL-derived tendon cells. An age stratification analysis found no effect on growth and gene expression. COL1 was found to be higher in males when compared to females (P ​< ​0.001), but otherwise no difference was seen in growth and gene expression in the gender analysis. No postbiopsy clinical complications were reported for either group., Conclusion: This study has shown that the growth and bioactivities of tendon cells from tendon biopsies for OrthoATI™ are not affected by tendon donor site and age., Level of Evidence: IV., Competing Interests: Declaration of competing interest MHZ & AW hold shares of Orthocell Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Background: Tendons are important dense fibrous structures connecting muscle to bone, and tendon stem cells (TDSCs) affect their repair and regeneration. The role of TDSC-derived exosomes (TDSC-Exos) is still being unexplored; therefore, this study aimed to investigate the protective effect of TDSC-Exos on tenocytes., Methods: The TDSCs and tenocytes were all derived from Sprague Dawley (SD) rats. The expression of positive and negative markers of TDSCs were detected by flow cytometry, and the multi-differentiation ability was also detected to identify TDSCs. Exos were derived from TDSCs using ultracentrifugation; furthermore, Exos enriched with microRNA(miR)-377-3p were generated from TDSCs stably overexpressing miR-377-3p after transfection, identified with transmission electron microscopy (TEM), western blot and PKH26 staining assay. Moreover, the cell functions of tenocytes were evaluated by MTT, EdU, transwell, and flow cytometry. Dual luciferase reporter and RNA pull-down assays were used to verify the binding sites of miR-337-3p and caspase3 (CASP3) predicted by Targetscan., Results: Exos (miR-337-3p) were taken up by tenocytes, and promoted the proliferation, migration, and invasion and suppressed the apoptosis of tenocytes in a dose-dependent manner. Bioinformatics analysis showed that CASP3 was a target of miR-377-3p, which was further verified by luciferase and RNA pull-down assays. Moreover, over-expressed CASP3 reversed the effects of Exos (miR-337-3p) on cell functions of tenocytes., Conclusions: Our findings suggest that Exos derived from miR-337-3p over-expressing TDSCs could potentially protect against tenocyte apoptosis by regulating CASP3. This novel therapeutic approach holds promise for the treatment of tendon injury, offering a glimmer of hope for improved patient outcomes., (© 2024. The Author(s).)

Inflammation is a driving force of tendinopathy. The oxidation of phospholipids by free radicals is a consequence of inflammatory reactions and is an important indicator of tissue damage. Here, we have studied the impact of oxidized phospholipids (OxPAPC) on the function of human tenocytes. We observed that treatment with OxPAPC did not alter the morphology, growth and capacity to produce collagen in healthy or diseased tenocytes. However, since OxPAPC is a known modulator of the function of immune cells, we analyzed whether OxPAPC-treated immune cells might influence the fate of tenocytes. Co-culture of tenocytes with immature, monocyte-derived dendritic cells treated with OxPAPC (Ox-DCs) was found to enhance the proliferation of tenocytes, particularly those from diseased tendons. Using transcriptional profiling of Ox-DCs, we identified amphiregulin (AREG), a ligand for EGFR, as a possible mediator of this proliferation enhancing effect, which we could confirm using recombinant AREG. Of note, diseased tenocytes were found to express higher levels of EGFR compared to tenocytes isolated from healthy donors and show a stronger proliferative response upon co-culture with Ox-DCs, as well as AREG treatment. In summary, we identify an AREG-EGFR axis as a mediator of a DC-tenocyte crosstalk, leading to increased tenocyte proliferation and possibly tendon regeneration.

A major problem after tendon injury is adhesion formation to the surrounding tissue leading to a limited range of motion. A viable strategy to reduce adhesion extent is the use of physical barriers that limit the contact between the tendon and the adjacent tissue. The purpose of this study was to fabricate an electrospun bilayered tube of hyaluronic acid/polyethylene oxide (HA/PEO) and biodegradable DegraPol® (DP) to improve the anti-adhesive effect of the implant in a rabbit Achilles tendon full laceration model compared to a pure DP tube. Additionally, the attachment of rabbit tenocytes on pure DP and HA/PEO containing scaffolds was tested and Scanning Electron Microscopy, Fourier-transform Infrared Spectroscopy, Differential Scanning Calorimetry, Water Contact Angle measurements, and testing of mechanical properties were used to characterize the scaffolds. In vivo assessment after three weeks showed that the implant containing a second HA/PEO layer significantly reduced adhesion extent reaching levels comparable to native tendons, compared with a pure DP implant that reduced adhesion formation only by 20 %. Tenocytes were able to attach to and migrate into every scaffold, but cell number was reduced over two weeks. Implants containing HA/PEO showed better mechanical properties than pure DP tubes and with the ability to entirely reduce adhesion extent makes this implant a promising candidate for clinical application in tendon repair., Competing Interests: Declaration of competing interest The authors declare that they do not have any competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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).)

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.)

Tissue fibrosis following tendon injury is a major clinical problem due to the increased risk of re-injury and limited treatment options; however, its mechanism remains unclear. Evidence suggests that insufficient resolution of inflammation contributes to fibrotic healing by disrupting tenocyte activity, with the NF-κB pathway being identified as a potential mediator. Equine embryonic stem cell (ESC) derived tenocytes may offer a potential cell-based therapy to improve tendon regeneration, but how they respond to an inflammatory environment is largely unknown. Our findings reveal for the first time that, unlike adult tenocytes, ESC-tenocytes are unaffected by IFN-γ, TNFα, and IL-1β stimulation; producing minimal changes to tendon-associated gene expression and generating 3-D collagen gel constructs indistinguishable from unstimulated controls. Inflammatory pathway analysis found these inflammatory cytokines failed to activate NF-κB in the ESC-tenocytes. However, NF-κB could be activated to induce changes in gene expression following stimulation with NF-κB pharmaceutical activators. Transcriptomic analysis revealed differences between cytokine and NF-κB signalling components between adult and ESC-tenocytes, which may contribute to the mechanism by which ESC-tenocytes escape inflammatory stimuli. Further investigation of these molecular mechanisms will help guide novel therapies to reduce fibrosis and encourage superior tendon healing., (© 2024. The Author(s).)

Lidocaine is the most frequently applied local infiltration anesthetic agent for treating tendinopathies. However, studies have discovered lidocaine to negatively affect tendon healing. In the current study, the molecular mechanisms and effects of lidocaine on tenocyte migration were evaluated. We treated tenocytes intrinsic to the Achilles tendons of Sprague-Dawley rats with lidocaine. The migration ability of cells was analyzed using electric cell-substrate impedance sensing (ECIS) and scratch wound assay. We then used a microscope to evaluate the cell spread. We assessed filamentous actin (F-actin) cytoskeleton formation through immunofluorescence staining. In addition, we used Western blot analysis to analyze the expression of phospho-focal adhesion kinase (FAK), FAK, phospho-paxillin, paxillin, and F-actin. We discovered that lidocaine had an inhibitory effect on the migration of tenocytes in the scratch wound assay and on the ECIS chip. Lidocaine treatment suppressed cell spreading and changed the cell morphology and F-actin distribution. Lidocaine reduced F-actin formation in the tenocyte during cell spreading; furthermore, it inhibited phospho-FAK, F-actin, and phospho-paxillin expression in the tenocytes. Our study revealed that lidocaine inhibits the spread and migration of tenocytes. The molecular mechanism potentially underlying this effect is downregulation of F-actin, phospho-FAK, and phospho-paxillin expression when cells are treated with lidocaine., (© 2023 Orthopaedic Research Society.)

We investigated how Interleukin 1 beta (IL-1β) impacts equine tenocyte function and global gene expression in vitro and determined if these effects could be rescued by pharmacologically inhibiting nuclear factor-κB (NF- K B) or interleukin 1 signalling. Equine superficial digital flexor tenocytes were cultured in three-dimensional (3D) collagen gels and stimulated with IL-1β for two-weeks, with gel contraction and interleukin 6 (IL6) measured throughout and transcriptomic analysis performed at day 14. The impact of three NF- K B inhibitors on gel contraction and IL6 secretion were measured in 3D culture, with NF- K B-P65 nuclear translocation by immunofluorescence and gene expression by qPCR measured in two-dimensional (2D) monolayer culture. In addition, daily 3D gel contraction and transcriptomic analysis was performed on interleukin 1 receptor antagonist-treated 3D gels at day 14. IL-1β increased NF- K B-P65 nuclear translocation in 2D culture and IL6 secretion in 3D culture, but reduced daily tenocyte 3D gel contraction and impacted > 2500 genes at day 14, with enrichment for NF- K B signaling. Administering direct pharmacological inhibitors of NF- K B did reduce NF- K B-P65 nuclear translocation, but had no effect on 3D gel contraction or IL6 secretion in the presence of IL-1β. However, IL1Ra restored 3D gel contraction and partially rescued global gene expression. Tenocyte 3D gel contraction and gene expression is adversely impacted by IL-1β which can only be rescued by blockade of interleukin 1 receptor, but not NF- K B, signalling., (© 2023. The Author(s).)

The myotendinous junction (MTJ) is a vulnerable region at the interface of skeletal muscle and tendon that forms an integrated mechanical unit. This study presents a technique for the spatially restrictive co-culture of human embryonic stem cell (hESC)-derived skeletal myocytes and primary tenocytes for two-dimensional modeling of the MTJ. Micropatterned lanes of extracellular matrix and a 2-well culture chamber define the initial regions of occupation. On day 1, both lines occupy less than 20 % of the initially vacant interstitial zone, referred to henceforth as the junction. Myocyte-tenocyte interdigitations are observed by day 7. Immunocytochemistry reveals enhanced organization and alignment of patterned myocyte and tenocyte features, as well as differential expression of multiple MTJ markers. On day 24, electrically stimulated junction myocytes demonstrate negative contractile strains, while positive tensile strains are exhibited by mechanically passive tenocytes at the junction. Unpatterned tenocytes distal to the junction experience significantly decreased strains in comparison to cells at the interface. Unpatterned myocytes have impaired organization and uncoordinated contractile behavior. These findings suggest that this platform is capable of inducing myocyte-tenocyte junction formation and mechanical coupling similar to the native MTJ, showing transduction of force across the cell-cell interface. STATEMENT OF SIGNIFICANCE: The myotendinous junction (MTJ) is an integrated structure that transduces force across the muscle-tendon boundary, making the region vulnerable to strain injury. Despite the clinical relevance, previous in vitro models of the MTJ lack the structure and mechanical accuracy of the native tissue and have difficulty transmitting force across the cell-cell interface. This study demonstrates an in vitro model of the MTJ, using spatially restrictive cues to inform human myocyte-tenocyte interactions and architecture. The model expressed MTJ markers and developed anisotropic myocyte-tenocyte integrations that resemble the native tissue and allow for force transduction from contracting myocytes to passive tenocyte regions. As such, this study presents a system capable of investigating development, injury, and pathology in the human MTJ., 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 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-β (TGFβ) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFβ signaling in tendon prompted us to utilize TGFβ1 to induce tendon-like structures in 3D tendon constructs. TGFβ1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFβ1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFβ1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization., (© 2024. The Author(s).)

Heterotopic ossification (HO) is a challenging condition that occurs after musculoskeletal injury and is characterized by the formation of bone in non-skeletal tissues. While the effect of HO on blood vessels is well established, little is known about its impact on lymphatic vessels. Here, we use a mouse model of traumatic HO to investigate the relationship between HO and lymphatic vessels. We show that injury triggers lymphangiogenesis at the injury site, which is associated with elevated vascular endothelial growth factor C (VEGF-C) levels. Through single-cell transcriptomic analyses, we identify mesenchymal progenitor cells and tenocytes as sources of Vegfc. We demonstrate by lineage tracing that Vegfc-expressing cells undergo osteochondral differentiation and contribute to the formation of HO. Last, we show that Vegfc haploinsufficiency results in a nearly 50% reduction in lymphangiogenesis and HO formation. These findings shed light on the complex mechanisms underlying HO formation and its impact on lymphatic vessels., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: The study objectives were to 1) determine the mesenchymal stem cell (MSC) surface expression of major histocompatibility complex (MHC) class I and transcriptome-wide gene expression changes following IL-1β + TGF-β2 dual licensing and 2) evaluate if IL-1β + TGF-β2 dual-licensed MSCs had a greater ability to positively modulate tenocyte function compared to naive MSCs., Sample: Equine bone marrow-derived MSCs from 6 donors and equine superficial digital flexor tenocytes from 3 donors., Methods: Experiments were performed in vitro. Flow cytometry and bulk RNA sequencing were utilized to determine naive and dual-licensed MSC phenotype and transcriptome-wide changes in gene expression. Conditioned media were generated from MSCs and utilized in tenocyte cell culture assays as a method to determine the effect of MSC paracrine factors on tenocyte function., Results: Dual-licensed MSCs have a reduced expression of MHC class I and exhibit enrichment in functional pathways associated with the extracellular matrix, cell signaling, and tissue development. Additionally, dual-licensed MSC-conditioned media significantly improved in vitro tenocyte migration and metabolism to a greater degree than naive MSC-conditioned media. In tenocytes exposed to IL-1β, dual-licensed conditioned media also positively modulated tenocyte gene expression., Clinical Relevance: Our data indicate that conditioned media containing paracrine factors secreted from dual-licensed MSCs significantly modulates in vitro tenocyte function, which may confer benefits in vivo to healing tendons following injury. Additionally, due to reduced MHC class I expression in dual-licensed MSCs, this technique may also provide an avenue to provide an effective "off-the-shelf" allogenic source of MSCs.

Objective: Tendinopathy is influenced by multiple factors, including chronic inflammation and aging. Senescent cells exhibit characteristics such as the secretion of matrix-degrading enzymes and pro-inflammatory cytokines, collectively known as senescence-associated secretory phenotypes (SASPs). Many of these SASP cytokines and enzymes are implicated in the pathogenesis of tendinopathy. MicroRNA-146a (miR-146a) blocks senescence by targeting interleukin-1β (IL-1β) receptor-associated kinase 4 (IRAK-4) and TNF receptor-associated factor 6 (TRAF6), thus inhibiting NF-κB activity. The aims of this study were to (1) investigate miR-146a expression in tendinopathic tendons and (2) evaluate the role of miR-146a in countering senescence and SASPs in tendinopathic tenocytes., Methods: MiR-146a expression was assessed in human long head biceps (LHB) and rat tendinopathic tendons by in situ hybridization. MiR-146a over-expression in rat primary tendinopathic tenocytes was achieved by lentiviral vector-mediated precursor miR-146a transfer (LVmiR-146a). Expression of various senescence-related markers was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunoblotting and immunofluorescence. MiR-146a expression showed a negative correlation with the severity of tendinopathy in human and rat tendinopathic tendons (p<0.001)., Results: Tendinopathic tenocyte transfectants overexpressing miR-146a exhibited downregulation of various senescence and SASP markers, as well as the target molecules IRAK-4 and TRAF6, and the inflammatory mediator phospho-NF-κB. Additionally, these cells showed enhanced nuclear staining of high mobility group box 1 (HMGB1) compared to LVmiR-scramble-transduced controls in response to IL-1β stimulation., Conclusions: We demonstrate that miR-146a expression is negatively correlated with the progression of tendinopathy. Moreover, its overexpression protects tendinopathic tenocytes from SASPs and senescence through the IRAK-4/TRAF6/NF-kB pathway.

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 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.)

Transforming growth factor-beta (TGF-β1) induces plasminogen activator inhibitor 1 (PAI-1) to effect fibrotic pathologies in several organs including tendon. Recent data implicated PAI-1 with inhibition of phosphatase and tensin homolog (PTEN) suggesting that PAI-1-induced adhesions involves phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (mTOR) signaling. Ergo, we investigated effects of TGF-β1, PAI-1, and mTOR signaling crosstalk on myofibroblast activation, senescence, and proliferation in primary flexor tenocytes from wild-type (WT) and PAI-1 knockout (KO) mice. PAI-1 deletion blunted TGF-β1-induced myofibroblast activation in murine flexor tenocytes and increased the gene expression of Mmp-2 to confer protective effects against fibrosis. While TGF-β1 significantly reduced phosphorylation of PTEN in WT cells, PAI-1 deletion rescued the activation of PTEN. Despite that, there were no differences in TGF-β1-induced activation of mTOR signaling (AKT, 4EBP1, and P70S6K) in WT or KO tenocytes. Phenotypic changes in distinct populations of WT or KO tenocytes exhibiting high or low mTOR activity were then examined. TGF-β1 increased alpha-smooth muscle actin abundance in WT cells exhibiting high mTOR activity, but this increase was blunted in KO cells exhibiting high 4EBP1 activity but not in cells exhibiting high S6 activity. DNA damage (γH2AX) was increased with TGF-β1 treatment in WT tenocytes but was blunted in KO cells exhibiting high mTOR activity. Increased mTOR activity enhanced proliferation (Ki67) in both WT and KO tenocytes. These findings point to a complex nexus of TGF-β1, PAI-1, and mTOR signaling in regulating proliferation, myofibroblast differentiation, and senescence in tenocytes, which could define therapeutic targets for chronic tendon adhesions and other fibrotic pathologies., (© 2023 Orthopaedic Research Society.)

Objective: To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture., Sample: Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14+ monocytes from 5 healthy horses ages 9-11 years, euthanized for reasons unrelated to musculoskeletal conditions., Methods: Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA., Results: Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged., Clinical Relevance: Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.

Tendon overuse injuries are common, but the processes that govern tendon response to mechanical load are not fully understood. A series of experiments of in vitro and in vivo experiments was devised to study to the relationship between mechanical stimuli and the matricellular protein Cellular Communication Network Factor 1 (CCN1) in tenocytes and tendons. First, human and murine tenocytes were subjected to cyclic uniaxial loading in order to evaluate changes in CCN1 gene expression as a response to mechanical stimuli. Then, baseline Ccn1 gene expression in different murine tendons (Achilles, patellar, forearm, and tail) was examined. Finally, changes in Ccn1 expression after in vivo unloading experiments were examined. It was found that CCN1 expression significantly increased in both human and murine tenocytes at 5 and 10% cyclical uniaxial strain, while 2.5% strain did not have any effect on CCN1 expression. At baseline, the Achilles, patellar, and forearm tendons had higher expression levels of Ccn1 as compared to tail tendons. Twenty-four hours of immobilization of the hind-limb resulted in a significant decrease in Ccn1 expression in both the Achilles and patellar tendons. In summary, CCN1 expression is up-regulated in tenocytes subjected to mechanical load and down-regulated by loss of mechanical load in tendons. These results show that CCN1 expression in tendons is at least partially regulated by mechanical stimuli., 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., (Published by Elsevier Inc.)

High monosaccharide levels are intimately associated with diabetes and impact tendon cells through inflammation and impairment in metabolic homeostasis. Experiments were designed to understand the responses elicited by cultured tenocytes under monosaccharide stress induced by hyperglycemia and hyperfructosemia. We simulated hyperglycemia and hyperfructosemia in vitro by treating tenocytes with media containing sublethal concentrations of glucose and fructose, respectively. Exposure of tenocytes to high glucose and high fructose altered the levels of IL-1β, IL-2, IL-6, IL10 and IL-17A. AMPK expression was increased in high-glucose and decreased in high-fructose groups. High fructose increased the level of IRS-1 compared with the control. Increased mitochondrial superoxide levels and compromised mitochondrial membrane integrity were exhibited by both the groups. The findings from the network analysis revealed many altered genes that are related to pathways for enzyme-linked receptor protein signaling, positive regulation of metabolic processes, transmembrane receptor tyrosine kinase pathway, insulin receptor signaling and regulation of cytokine production. Overall, the data suggest that the tenocytes under high monosaccharide levels exhibit survival responses by altering the expression status of cytokines and metabolic mediators that are involved in the underlying pathogenesis of tendinopathy., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Tendinopathy, a prevalent overuse injury, lacks effective treatment options, leading to a significant impact on quality of life and socioeconomic burden. Mesenchymal stem/stromal cells (MSCs) and their secretome, including conditioned medium (CM) and extracellular vesicles (EVs), have shown promise in tissue regeneration and immunomodulation. However, it remains unclear which components of the secretome contribute to their therapeutic effects. This study aimed to compare the efficacy of CM, EVs, and the soluble protein fraction (PF) in treating inflamed tenocytes. CM exhibited the highest protein and particle concentrations, followed by PF and EVs. Inflammation significantly altered gene expression in tenocytes, with CM showing the most distinct separation from the inflamed control group. Treatment with CM resulted in the most significant differential gene expression, with both upregulated and downregulated genes related to inflammation and tissue regeneration. EV treatment also demonstrated a therapeutic effect, albeit to a lesser extent. These findings suggest that CM holds superior therapeutic efficacy compared with its EV fraction alone, emphasizing the importance of the complete secretome in tendon injury treatment.

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).)

Objectives: Basic calcium phosphate (BCP) crystals contribute to several syndromes associated with tendon disease, including acute calcific tendinitis and Milwaukee shoulder syndrome. Interactions between BCP crystals and tenocytes (tendon cells) may contribute to these clinical syndromes. This study aimed to determine the direct effects of BCP crystals on tenocyte function and viability., Methods: In vitro assays were used to assess changes in human tenocytes cultured with BCP crystals. Real-time PCR was used to determine changes in the expression of tendon-related genes and extracellular matrix remodelling enzymes (MMPs; a disintegrin and metalloproteases, ADAMTS; and tissue inhibitor of metalloproteinases, TIMPs). ELISA was used to measure protein concentrations in tenocyte supernatants. MTT and alamarBlue™ assays were used to determine changes in cell viability., Results: BCP crystals upregulated tenocyte gene expression of MMP-1, MMP-3, ADAMTS-4 and TIMP-1 after 24 h. Time-course experiments showed expression peaked at 8 h for TIMP-1 and 48 h for MMP-1 and ADAMTS-4. Cyclooxygenase (COX)-1 gene expression was upregulated after 48 h. Tenocytes did not alter expression of scleraxis and tendon collagens, and expression of pro-inflammatory cytokines was not induced with BCP crystals. BCP crystals increased tenocyte release of prostaglandin E2 (PGE2) and MMP-1 protein after 24 h. However, neither COX-1 inhibition nor COX-2 inhibition led to consistent change in BCP crystal-induced tenocyte gene expression of extracellular matrix remodelling enzymes. BCP crystals had no effect on tenocyte viability., Conclusion: BCP crystals induce extracellular matrix remodelling enzymes, but not inflammatory cytokines, in tenocytes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Rotator cuff Tear (RCT) causes a lot of inconvenience for patients. In most cases, RCT injury does not heal back to bone after repair, and there is a high chance of retearing. Therefore, there is a need to explore more effective targeted therapies. Bone mesenchymal stem cell-derived exosome (BMSCs-Exo) has been proved to be beneficial to the proliferation of tendon cells, but its specific mechanism remains to be further explored., Methods: BMSCs-Exo was isolated and identified by detecting the specific markers using flow cytometry and western blot assays. qRT-PCR and western blot were utilized to determine the gene or protein expressions, respectively. Cell proliferation, and migration in tenocytes were measured by CCK8, EdU and transwell assays. The interaction between miR-29a and FABP3 was analyzed using dual-luciferase reporter assay., Results: Our findings demonstrated that miR-29a was expressed in BMSCs-Exo and could be significantly enriched after TGF-β1 treatment. Moreover, TGF-β1-modified BMSCs-Exo co-cultured could promote the proliferation, migration and fibrosis of tenocytes by carrying miR-29a. Upon miR-29a was reduced in BMSCs-Exo, the regulatory roles of BMSCs-Exo on tenocytes were reversed. Mechanistically, miR-29a negatively regulated FABP3 via interaction with its 3'-UTR. Enforced expression of FABP3 could reverse the modulation of exosomal miR-29a in tenocytes., Conclusion: Exosomal miR-29a derived from TGF-β1-modified BMSCs facilitated the proliferation, migration and fibrosis of tenocytes through targeting FABP3., 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 © 2022 Elsevier Ltd. All rights reserved.)

Primary tenocytes rapidly undergo senescence and a phenotypic drift upon in vitro monolayer culture, which limits tendon research. The Ink4a/Arf locus encodes the proteins p16 Ink4a/Arf and p14 ARF (p19 ARF in mice) that regulate cell cycle progression and senescence. We here established an immortalized cell line using tenocytes isolated from Ink4a/Arf deficient mice (Ink4a/Arf -/- ). These cells were investigated at three distinct time points, at low (2-5), intermediate (14-17) and high (35-44) passages. Wild-type cells at low passage (2-5) served as controls. Ink4a/Arf -/- tenocytes at all stages were comparable to wild-type cells regarding morphology, expression of tenogeneic genes (collagen type 1, 3 and 5, Scleraxis, Tenomodulin and Tenascin-C), and surface markers (CD29, CD44 and CD105) and form 3D tendon-like structures. Importantly, Ink4a/Arf -/- tenocytes maintained their phenotypic features and proliferation potential in culture for more than 40 passages and also following freeze-thaw cycles. In contrast, wild-type tenocytes underwent senescence starting in passage 6. These data define Ink4a/Arf -/- tenocytes as novel tool for in vitro tendon research and as valuable in vitro alternative to animal experiments., (© 2023. The Author(s).)

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that regulates tendon development and maturation. Our previous study indicated that mechanical stretch could induce Tnmd expression to promote tenocyte migration, associated with reinforcement of fibrous actin (F-actin) stress fibers and chromatin decondensation. However, the detailed molecular mechanisms of this processes are far from clear. Activation of mitogen-activated protein kinase (MAPK) signaling occurs in response to various extracellular stimuli and controls a large number of fundamental cellular processes. The present study we investigated the influence of MAPK signaling on mechanical stretch-induced Tnmd expression and its action way. Expression and activities of extracellular signal-related kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK (p38) were determined by Western blot. Cell migration was detected by Transwell assay. Immunofluorescence staining was used to detect F-actin stress fibers. Nuclear chromatin decondensation was detected by in situ DNaseI sensitivity assay. It was found that mechanical stretch promoted Tnmd expression by activating ERK1/2, JNK and p38 signaling. The inhibition of the ERK1/2, JNK or p38 repressed mechanical stretch-promoted tenocyte migration and mechanical stretch-induced reinforcement of F-actin stress fibers. However, only ERK1/2 and p38 inhibitor could repress mechanical stretch-induced chromatin decondensation, and the JNK inhibitor had no significant effect. Moreover, latrunculin (Lat A), the most widely used reagent to depolymerize actin filaments, could inhibit the stretch-induced chromatin decondensation. Taken together, our findings elucidated a molecular pathway by which a mechanical signal is transduced via activation of MAPK signaling to influence reinforcement of F-actin stress fibers and chromatin decondensation, which could further lead Tnmd expression to promote tenocyte migration., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We generated a novel tetracycline-inducible transgenic mouse line with the tendon-specific expression of a series of tendon-critical transcription factors. Primary tenocytes derived from this mouse line consistently expressed green fluorescent protein reporter transcription factors in response to doxycycline. The tenocytes maintained their tendon cell properties for a longer time after the transient induction in the absence of growth factors and mechanical stress. Four key transcription factors for tendon development and the green fluorescent protein reporter were linked with different viral 2A self-cleaving peptides. They were expressed under the control of the tet-responsive element. In combination with the expression of BFP, which reports on the tendon-specific collagen I, and mScarlet, which reports on the tendon-specific transcription factor Scleraxis (Scx), we observed the more extended maintenance of the tendon cell identity of in vitro cultured tendon cells and Achilles tendon explants. This means that the Scleraxis bHLH transcription factor (Scx), mohawk homeobox (Mkx), early growth response 1 (Egr1) and early growth response 2 (Egr2) contributed to the maintenance of tenocytes' identity in vitro, providing a new model for studying extracellular matrix alterations and identifying alternative biomaterials in vitro.

Purpose: Mechanical stimuli are essential for the maintenance of tendon tissue homeostasis. The study aims to elucidate the mechanobiological mechanisms underlying the maintenance of tenocyte homeostasis by cyclic mechanical stretch under high-glucose (HG) condition., Materials and Methods: Primary tenocytes were isolated from rat Achilles tendon and 2D-cultured under HG condition. The in vitro effects of a single bout, 2-h cyclic biaxial stretch session (1 Hz, 8%) on primary rat tenocytes were explored through Flexcell system. Cell viability, tenogenic gene expression, intracellular calcium concentration, focal adhesion kinase (FAK) expression, and signaling pathway activation were analyzed in tenocytes with or without mechanical stretch., Results: Mechanical stretch increased tenocyte proliferation and upregulated early growth response protein 1 (Egr1) expression. An increase in intracellular calcium was observed after 30 min of stretching. Mechanical stretch phosphorylated FAK, calmodulin-dependent protein kinase kinase 2 (CaMKK2), and 5' adenosine monophosphate-activated protein kinase (AMPK) in a time-dependent manner, and these effects were abrogated after blocking intracellular calcium. Inhibition of FAK, CaMKK2, and AMPK downregulated the expression of Egr1. In addition, mechanical stretch reinforced cytoskeletal organization via calcium (Ca2+)/FAK signaling., Conclusions: Our study demonstrated that mechanical stretch-induced calcium influx activated CaMKK2/AMPK signaling and FAK-cytoskeleton reorganization, thereby promoting the expression of Egr1, which may help maintain tendon cell characteristics and homeostasis in the context of diabetic tendinopathy.

The combination of cross-linked hyaluronate (cHA) and corticosteroid showed more rapid pain or functional improvement in knee osteoarthritis and adhesive capsulitis. However, rare evidence of this combination in treating tendinopathy has been reported. We hypothesized that the specific formulations of cHA and dexamethasone (DEX) conferred amelioration of tendinopathy via anti-apoptosis and anti-senescence. In this controlled laboratory study, primary tenocytes from the human tendinopathic long head of biceps were treated with three cHA formulations (cHA:linealized HA = 80:20, 50:50, and 20:80) + DEX with or without IL-1β stimulation. Cell viability, inflammatory cytokines, tendon-related proliferation markers, matrix metalloproteinases (MMPs), senescent markers, and apoptosis were examined. The in vivo therapeutic effects of the selected cHA + DEX combinations were evaluated in a collagenase-induced rat patellar tendinopathy model. The expression levels of inflammatory mediators, including IL-1β, IL-6, COX-2, MMP-1, and MMP-3 were significantly reduced in all cHA + DEX-treated tenocytes (p < 0.05, all). The cHA (50:50) + DEX and cHA (20:80) + DEX combinations protected tenocytes from cytotoxicity, senescence, and apoptosis induced by DEX in either IL-1β stimulation or none. Furthermore, the two combinations significantly improved the rat experimental tendinopathy by reducing ultrasound feature scores and histological scores as well as the levels of apoptosis, senescence, and senescence-associated secretory phenotypes (p < 0.05, all). We identified two specific cHA formulations (cHA (50:50) and cHA (20:80)) + DEX that could ameliorate tendinopathy through anti-senescence and -apoptosis without cytotoxicity. This study provides a possible approach to treating tendinopathy using the combination of two well-known agents.

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.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3&#95;PAPPA, POSTN&#95;SCX, DCN&#95;LUM and ITGA7&#95;NES. Surface proteomics found that PTX3&#95;PAPPA cells were CD10+CD26+CD54+. ITGA7&#95;NES cells were CD146+ and POSTN&#95;SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN&#95;LUM, POSTN&#95;SCX and PTX3&#95; PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.

Background: Studies in our laboratory have demonstrated mitochondrial dysfunction in human and animal models of supraspinatus tendinopathy. SS-31 (elamipretide) has been reported to improve mitochondrial function and to be effective in clinical trials for several diseases. The potential of SS-31 in treating tendinopathy has not been explored., Hypothesis: SS-31 would improve mitochondrial function in human tenocytes sampled from patients with tendinopathy., Study Design: Controlled laboratory study., Methods: Healthy tenocytes were obtained from normal hamstring tendon biopsy specimens in 9 patients undergoing anterior cruciate ligament reconstruction, and tenocytes were collected from degenerative supraspinatus tendon biopsy specimens in 9 patients undergoing rotator cuff repair. Tenocytes were cultured, used at passage 1, and assigned to 4 groups: healthy tenocytes, healthy tenocytes with 1μM SS-31 treatment for 72 hours, degenerative tenocytes, and degenerative tenocytes with 1μM SS-31 treatment for 72 hours. The outcomes included measurements of mitochondrial potential, mitochondrial morphology by transmission electron microscopy imaging, reactive oxygen species and superoxidative dismutase activity, gene expression, and cell viability., Results: An increase in the cell fraction with depolarized mitochondria was found in degenerative tenocytes ( P = .014), followed by a decrease after SS-31 treatment ( P = .018). Transmission electron microscopy images demonstrated morphological changes with a decreased number and size of mitochondria per cell in the degenerative tenocytes ( P = .018) and with improvement after SS-31 treatment. There was no significant difference in the level of reactive oxygen species between healthy and degenerative tenocytes in culture, but superoxidative dismutase activity was significantly decreased in the degenerative group ( P = .006), which then increased after SS-31 treatment ( P = .012). These findings suggested that mitochondrial dysfunction may be reversed by SS-31 treatment. The gene expression of matrix metalloproteinase-1 (matrix remodeling, P = .029) and fatty acid-binding protein 4 (fatty infiltration, P = .046) was significantly upregulated in the degenerative tenocytes and reduced by SS-31 treatment ( P = .048; P = .007). Gene expression for hypoxia-inducible factor1 α and the proapoptotic regulator Bcl-2-associated X protein was increased in the degenerative tenocytes. There was a significant decrease in cell viability in degenerative tenocytes as compared with the healthy tenocytes, with small improvement after treatment with SS-31., Conclusion: There are changes in mitochondrial structure and function in tenocytes derived from degenerative tendons, and SS-31, as a mitochondrial protectant, could improve mitochondrial function and promote the healing of tendinopathy., Clinical Relevance: Mitochondrial dysfunction appears to play a role in the development of tendinopathy, and SS-31, as a mitochondrial protective agent, may be a therapeutic agent in the treatment of tendinopathy.

(1) Background: Surgical tendon repair often leads to adhesion formation, leading to joint stiffness and a reduced range of motion. Tubular implants set around sutured tendons might help to reduce peritendinous adhesions. The lubricant hyaluronic acid (HA) is a viable option for optimizing such tubes with the goal of further enhancing the anti-adhesive effect. As the implant degrades over time and diffusion is presumed, the impact of HA on tendon cells is important to know. (2) Methods: A culture medium of rabbit Achilles tenocytes was supplemented with high-molecular-weight (HMW) HA and the growth curves of the cells were assessed. Additionally, after 3, 7 and 14 days, the gene expression of several markers was analyzed for matrix assembly, tendon differentiation, fibrosis, proliferation, matrix remodeling, pro-inflammation and resolution. (3) Results: The addition of HA decreased matrix marker genes, downregulated the fibrosis marker α-SMA for a short time and slightly increased the matrix-remodeling gene MMP-2. Of the pro-inflammatory marker genes, only IL-6 was significantly upregulated. IL-6 has to be kept in check, although IL-6 is also needed for a proper initial inflammation and efficient resolution. (4) Conclusions: The observed effects in vitro support the intended anti-adhesion effect and therefore, the use of HMW HA is promising as a biodegradable implant for tendon repair.

Mesenchymal Stem Cells (MSCs) are important in regenerative medicine and tissue engineering and will be a very sensible choice for repair and regeneration of tendon. New biological practices, such as cellular therapy using stem cells, are promising for facilitating or expediting tendon therapy. Before using these cells clinically, it is best to check and confirm the optimal conditions for differentiation of these cells in the laboratory. Hence, in the present study, the impacts of PDGF-BB and GDF-6 supplementation on adipose-derived MSCs (ASCs) culture were studied. The frozen ASC were recovered and expanded in basic culture medium (DMEM with 10%FBS). The cells after passage five (P5) were treated with basic medium containing L-Prolin, Ascorbic Acid and only PDGF-BB or GDF-6 (20 ng/ml) or both of them (mix) as 3 groups for 14 days to investigate efficiency of ASCs differentiation towards tenocytes. The cells culturing in basic medium were used as control group. To validate tenogenic differentiation, H&E and Sirius Red staining were used to assess cell morphology and collagen production, respectively. In addition, mRNA levels of collagen I and III, Scleraxis and Tenomodulin as tenogenic markers were analyzed using qPCR. In all test groups, cells appeared slenderer, elongated cytoplasmic attributes compared to the control cells. The intensity of Sirius Red staining was significantly higher in GDF-6, PDGF-BB alone, than in group without supplements. The optical density was higher in the GDF-6 than PDGF-BB and mix-group. QPCR results showed that Col I and III gene expression was increased in all groups compared to the control. SCX expression was significantly increased only in the PDGF-BB group. TNMD mRNA expression was not significant among groups. In this study, we have corroborated that human ASCs are reactionary to tenogenic induction by GDF-6 and PDGF-BB alone or in combination. These outcomes will help greater insight into GDF-6 and PDGF-BB driven tenogenesis of ASCs and new directions of discovery in the design of ASC-based treatments for tendon healing., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

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.)

Background: The production of inflammatory mediators is critical for tenocytes proliferation and migration, which play an important role in rotator cuff injury repair and regulation of collagen. MicroRNA (miRNA)-205 (miR-205) promotes the secretion of inflammatory factors. The mechanism of the tenocytes regulation by miR-205 remains unknown. In this paper, we showed that miR-205 can regulate the proliferation, migration and fibrosis of tenocytes., Objectives: To investigate the function and mechanism of miR-205/MeCP2 pathway on the proliferation, migration and fibrosis of rotator cuff tenocytes, in order to provide a new perspective on the repair of rotator cuff tear injury., Material and Methods: The tenocytes were collected under sterile conditions from the Achilles tendons of Sprague Dawley (SD) rats (weighing 150-200 g). The cells of passages 2-4 were used for the following experiments. All miRNA and vectors were transfected with Lipofectamine 2000. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR), Cell Counting Kit-8 (CCK-8) assay, luciferase reporter assay, and migration assay were performed. Then, immunoblotting analysis and statistical analysis were conducted., Results: The CCK-8 and migration assay revealed that miR-205 inhibition resulted in increased tenocytes proliferation, migration and fibrosis. The miR-205 reduced the mRNA and protein expression levels of MECP2, which is involved in cell proliferation and migration of tenocytes. The miR-205 inhibited luciferase intensity under the control of the 3'UTRs of MECP2., Conclusions: The inhibition of MECP2 reversed the effect of miR-205 inhibitor on tenocytes, including the proliferation and migration of tenocytes, indicating that miR-205 may be valuable in miRNA-based therapies for rotator cuff injury.

Background: Tendon derived stem cells (TDSCs) have proven to be effective in tendon repair by secreting paracrine factors, which modulate the function of resident cells and inflammatory process. Exosomes, which are secreted from cells to mediate intercellular communication, may be used to treat tendon injuries. Here, we aimed to determine the effects of exosomes from TDSCs (TDSC-Exos) on tendon repair and to explore the underlying mechanism by investigating the role of microRNAs (miRNAs)., Methods: TDSC-Exos were isolated from TDSC conditioned medium. In vitro studies were performed to investigate the effects of TDSC-Exos on the proliferation, migration, cytoprotection, collagen production and tendon-specific markers expression in tenocytes. In order to determine the therapeutic effects of TDSC-Exos in vivo, we used a scaffold of photopolymerizable hyaluronic acid (p-HA) loaded with TDSC-Exos (pHA-TDSC-Exos) to treat tendon defects in the rat model. Subsequently, RNA sequencing and bioinformatic analyses were used to screen for enriched miRNAs in TDSC-Exos and predict target genes. The miRNA-target transcript interaction was confirmed by a dual-luciferase reporter assay system. In order to determine the role of candidate miRNA and its target gene in TDSC-Exos-regulated tendon repair, miRNA mimic and inhibitor were transfected into tenocytes to evaluate cell proliferation and migration., Results: Treatment with TDSC-Exos promoted proliferation, migration, type I collagen production and tendon-specific markers expression in tenocytes, and also protected tenocytes from oxidative stress and serum deprivation. The scaffold of pHA-TDSC-Exos could sever as a sustained release system to treat the rat model of tendon defects. In vivo study showed that TDSC-Exos promoted early healing of injured tendons. Rats treated with TDSC-Exos had better fiber arrangement and histological scores at the injury site. Besides, the injured tendons treated with TDSC-Exos had better performance in the biomechanical testing. Therefore, the pHA-TDSC-Exos scaffold proved to facilitate tendon repair in the rat model. miR-144-3p was enriched in TDSC-Exos and promoted tenocyte proliferation and migration via targeting AT-rich interactive domain 1A (ARID1A)., Conclusions: TDSC-Exos enhanced tenon repair through miR-144-3p-regulated tenocyte proliferation and migration. These results suggest that TDSC-Exos can serve as a promising strategy to treat tendon injuries., (© 2022. The Author(s).)

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.

It is known that mechanical loading of muscles increases the strength of healing tendon tissue, but the mechanism involved remains elusive. We hypothesized that the secretome from myoblasts in co-culture with tenocytes affects tenocyte migration, cell phenotype, and collagen (Col) production and that the effect is dependent on different types of mechanical loading of myoblasts. To test this, we used an in vitro indirect transwell co-culture system. Myoblasts were mechanically loaded using the FlexCell ® Tension system. Tenocyte cell migration, proliferation, apoptosis, collagen production, and several tenocyte markers were measured. The secretome from myoblasts decreased the Col I/III ratio and increased the expression of tenocyte specific markers as compared with tenocytes cultured alone. The secretome from statically loaded myoblasts significantly enhanced tenocyte migration and Col I/III ratio as compared with dynamic loading and controls. In addition, the secretome from statically loaded myoblasts induced tenocytes towards a myofibroblast-like phenotype. Taken together, these results demonstrate that the secretome from statically loaded myoblasts has a profound influence on tenocytes, affecting parameters that are related to the tendon healing process.

One great challenge in surgical tendon repair is the minimization of peritendinous adhesions. An electrospun tube can serve as a physical barrier around a conventionally sutured tendon. Six New Zealand White rabbits had one Achilles tendon fully transsected and sutured by a 4-strand suture. Another six rabbits had the same treatment, but with the additional electrospun DegraPol tube set around the sutured tendon. The adhesion formation to the surrounding tissue was investigated 12 weeks post-operation. Moreover, inflammation-related protease-activated receptor-2 (PAR-2) protein expression was assessed. Finally, rabbit Achilles tenocyte cultures were exposed to platelet-derived growth factor-BB (PDGF-BB), which mimicks the tendon healing environment, where PAR-2 gene expression was assessed as well as immunofluorescent staining intensity for F-actin and α-tubulin, respectively. At 12 weeks post-operation, the partially degraded DegraPol tube exhibited significantly lower adhesion formation (- 20%). PAR-2 protein expression was similar for time points 3 and 6 weeks, but increased at 12 weeks post-operation. In vitro cell culture experiments showed a significantly higher PAR-2 gene expression on day 3 after exposure to PDGF-BB, but not on day 7. The cytoskeleton of the tenocytes changed upon PDGF-BB stimulation, with signs of reorganization, and significantly decreased F-actin intensity. An electrospun DegraPol tube significantly reduces adhesion up to twelve weeks post-operation. At this time point, the tube is partially degraded, and a slight PAR-2 increase was detected in the DP treated tendons, which might however arise from particles of degrading DegraPol that were stained dark brown. PAR-2 gene expression in rabbit tenocytes reveals sensitivity at around day 10 after injury., (© 2021. The Author(s).)

Healing of ruptured tendons remains a clinical challenge because of its slow progress and relatively weak mechanical force at an early stage. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have therapeutic potential for tissue regeneration. In this study, we isolated EVs from adipose-derived stem cells (ADSCs) and evaluated their ability to promote tendon regeneration. Our results indicated that ADSC-EVs significantly enhanced the proliferation and migration of tenocytes in vitro. To further study the roles of ADSC-EVs in tendon regeneration, ADSC-EVs were used in Achilles tendon repair in rabbits. The mechanical strength, histology, and protein expression in the injured tendon tissues significantly improved 4 weeks after ADSC-EV treatment. Decorin and biglycan were significantly upregulated in comparison to the untreated controls. In summary, ADSC-EVs stimulated the proliferation and migration of tenocytes and improved the mechanical strength of repaired tendons, suggesting that ADSC-EV treatment is a potential highly potent therapeutic strategy for tendon injuries.