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To date, the cell and molecular mechanisms regulating tendon healing are poorly understood. Here, we establish a novel model of tendon regeneration using neonatal mice and show that neonates heal via formation of a ‘neo-tendon’ that differentiates along the tendon specific lineage with functional restoration of gait and mechanical properties. In contrast, adults heal via fibrovascular scar, aberrant differentiation toward cartilage and bone, with persistently impaired function. Lineage tracing identified intrinsic recruitment of Scx-lineage cells as a key cellular mechanism of neonatal healing that is absent in adults. Instead, adult Scx-lineage tenocytes are not recruited into the defect but transdifferentiate into ectopic cartilage; in the absence of tenogenic cells, extrinsic αSMA-expressing cells persist to form a permanent scar. Collectively, these results establish an exciting model of tendon regeneration and uncover a novel cellular mechanism underlying regenerative vs non-regenerative tendon healing.

Tendinopathy accounts for over 30% of primary care consultations and represents a growing healthcare challenge in an active and increasingly ageing population. Recognising critical cells involved in tendinopathy is essential in developing therapeutics to meet this challenge. Tendon cells are heterogenous and sparsely distributed in a dense collagen matrix; limiting previous methods to investigate cell characteristics ex vivo. We applied next generation CITE-sequencing; combining surface proteomics with in-depth, unbiased gene expression analysis of > 6400 single cells ex vivo from 11 chronically tendinopathic and 8 healthy human tendons. Immunohistochemistry validated the single cell findings. For the first time we show that human tendon harbours at least five distinct COL1A1/2 expressing tenocyte populations in addition to endothelial cells, T-cells, and monocytes. These consist of KRT7/SCX + cells expressing microfibril associated genes, PTX3 + cells co-expressing high levels of pro-inflammatory markers, APOD + fibro–adipogenic progenitors, TPPP3/PRG4 + chondrogenic cells, and ITGA7 + smooth muscle-mesenchymal cells. Surface proteomic analysis identified markers by which these sub-classes could be isolated and targeted in future. Chronic tendinopathy was associated with increased expression of pro-inflammatory markers PTX3 , CXCL1, CXCL6, CXCL8, and PDPN by microfibril associated tenocytes. Diseased endothelium had increased expression of chemokine and alarmin genes including IL33.

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.

Tendinopathies are a significant challenge in musculoskeletal medicine, with current treatments showing variable efficacy. Electromagnetic transduction therapy (EMTT) has emerged as a promising therapeutic approach, but its biological effects on tendon cells remain largely unexplored. Here, we investigated the effects of EMTT on primary cultured human tenocytes’ behavior and functions in vitro, focusing on cellular responses, senescence-related pathways, and molecular mechanisms. Primary cultures of human tenocytes were established from semitendinosus tendon biopsies of patients undergoing anterior cruciate ligament (ACL) reconstruction (n = 6, males aged 17–37 years). Cells were exposed to EMTT at different intensities (40 and 80 mT) and impulse numbers (1000–10,500). Cell viability (MTT assay), proliferation (Ki67), senescence markers (CDKN2a/INK4a), migration (scratch test), cytoskeleton organization (immunofluorescence), and gene expression (RT-PCR) were analyzed. A 40 mT exposure elicited minimal effects, whereas 80 mT treatments induced significant cellular responses. Repeated 80 mT exposure demonstrated a dual effect: despite a moderate decrease in overall cell vitality, increased Ki67 expression (+7%, p ≤ 0.05) and significant downregulation of senescence marker CDKN2a/INK4a were observed, suggesting potential senolytic-like activity. EMTT significantly enhanced cell migration (p < 0.001) and triggered cytoskeletal remodeling, with amplified stress fiber formation and paxillin redistribution. Molecular analysis revealed upregulation of tenogenic markers (Scleraxis, Tenomodulin) and enhanced Collagen I and III expressions, particularly with treatments at 80 mT, indicating improved matrix remodeling capacity. EMTT significantly promotes tenocyte proliferation, migration, and matrix production, while simultaneously exhibiting senolytic-like effects through downregulation of senescence-associated markers. These results support EMTT as a promising therapeutic approach for the management of tendinopathies through multiple regenerative mechanisms, though further studies are needed to validate these effects in vivo.

Mechanical forces between cells and extracellular matrix (ECM) influence cell shape and function. Tendons are ECM-rich tissues connecting muscles with bones that bear extreme tensional force. Analysis of transgenic zebrafish expressing mCherry driven by the tendon determinant scleraxis reveals that tendon fibroblasts (tenocytes) extend arrays of microtubule-rich projections at the onset of muscle contraction. In the trunk, these form a dense curtain along the myotendinous junctions at somite boundaries, perpendicular to myofibers, suggesting a role as force sensors to control ECM production and tendon strength. Paralysis or destabilization of microtubules reduces projection length and surrounding ECM, both of which are rescued by muscle stimulation. Paralysis also reduces SMAD3 phosphorylation in tenocytes and chemical inhibition of TGFβ signaling shortens tenocyte projections. These results suggest that TGFβ, released in response to force, acts on tenocytes to alter their morphology and ECM production, revealing a feedback mechanism by which tendons adapt to tension. Tendons – the fibrous structures that attach muscles to bones – must withstand some of the strongest forces in the body. Little is known about how tendons develop or adapt to withstand these forces. Studies have shown that muscles respond actively to force, as seen during exercise. Do tendons respond in similar ways? Tendons consist of collagen fibers surrounded by a ‘matrix’ of proteins. Also embedded in the matrix are specialized cells called tenocytes, which regulate the production of the different components of the tendon. A genetic modification allows tenocytes to be tracked using a fluorescent gene product that can be viewed using a microscope. Subramanian et al. have now used this technique in zebrafish to watch how the behaviors of the tenocytes change in response to forces applied to the tendon. Subramanian et al. show that at the start of muscle contraction, tenocytes put forth long projections from their cell bodies that extend perpendicular to the muscle fibers. This suggests that the projections act as force sensors. Consistent with this idea, paralyzing the muscle causes the projections to shrink. This shrinkage correlates with changes in how the tendon matrix proteins are organized. Further investigation reveals a force-responsive signaling pathway in the tenocytes that controls how these cells grow and produce key tendon matrix proteins. Subramanian et al. believe this pathway is central to how tendons adapt to the forces applied during muscle contraction. A better knowledge of how force affects tendon structure could ultimately help to improve treatments for tendon injuries and tendon atrophy. In particular, understanding how force affects how tenocytes develop could help researchers to develop new ways to regenerate and repair tendons.

Tendon ruptures have recently reached incidences of 18–35 cases/100,000 and often lead to adhesion formation during healing. Furthermore, scar formation may result in inferior biomechanics and often leads to re-ruptures. To address these problems, we cultivated rabbit adipose-derived stem cells in a co-culture with rabbit Achilles tenocytes and harvested their secretome. Following a cell-free approach, we incorporated such secretome into an electrospun tube via emulsion electrospinning. These novel implants were characterized by SEM, the WCA, and FTIR. Then, they were implanted in the rabbit Achilles tendon full transection model with an additional injection of secretome, and the adhesion extent as well as the biomechanics of extracted tendons were assessed three weeks postoperatively. The fiber thickness was around 3–5 μm, the pore size 11–13 μm, and the tube wall thickness approximately 265 μm. The WCA indicated slightly hydrophilic surfaces in the secretome-containing layer, with values of 80–90°. In vivo experiments revealed a significant reduction in adhesion formation (−22%) when secretome-treated tendons were compared to DegraPol® (DP) tube-treated tendons (no secretome). Furthermore, the cross-sectional area was significantly smaller in secretome-treated tendons compared to DP tube-treated ones (−32%). The peak load and stiffness of secretome-treated tendons were not significantly different from native tendons, while tendons treated with pure DP tubes exhibited significantly lower values. We concluded that secretome treatment supports tendon healing, with anti-adhesion effects and improved biomechanics at 3 weeks, making this approach interesting for clinical application.

Tendon ruptures and tendinopathies represent a major part of musculoskeletal injuries. Due to the hypovascular and hypocellular nature of tendons, the natural healing capacity is slow and limited. Cell-free approaches for tendon injuries are being investigated as the next generation of therapeutic treatments. The aim of this study was to compare the proteomic profiles and biological activities of two different secretomes, obtained from New Zealand white rabbit adipose-tissue-derived mesenchymal stem cells (ADSCs) or a 3:1 mixed culture of ADSCs and rabbit tenocytes. The secretomes were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) and their functional properties, such as gene expression, migration and angiogenesis, were investigated in vitro in rabbit tenocytes and in ovo using the chicken chorioallantoic membrane (CAM) assay after stimulation with secretomes or medium control. Both secretomes had a positive effect on angiogenesis and showed similar changes in relative gene expression levels associated with extracellular matrix (ECM) remodeling. Proteomic data showed that the two secretomes were clearly distinguishable, with 182 proteins significantly differentially expressed. The ADSC secretome was more effective in enhancing tenocyte migration under both healthy and inflammatory conditions. In the upregulated protein fraction of the mixed secretome, the tendon-related protein biglycan (BGN) and tenascin C (TNC) were increased. Based on our results, the mixed secretome shows great potential for promoting tendon healing as its composition is more effective in enhancing ECM-related processes and tendon development than the secretome of ADSCs.

Studies of cell fate focus on specification, but little is known about maintenance of the differentiated state. In this study, we find that the mouse tendon cell fate requires continuous maintenance in vivo and identify an essential role for TGFβ signaling in maintenance of the tendon cell fate. To examine the role of TGFβ signaling in tenocyte function the TGFβ type II receptor (Tgfbr2) was targeted in the Scleraxis-expressing cell lineage using the ScxCre deletor. Tendon development was not disrupted in mutant embryos, but shortly after birth tenocytes lost differentiation markers and reverted to a more stem/progenitor state. Viral reintroduction of Tgfbr2 to mutants prevented and even rescued tenocyte dedifferentiation suggesting a continuous and cell autonomous role for TGFβ signaling in cell fate maintenance. These results uncover the critical importance of molecular pathways that maintain the differentiated cell fate and a key role for TGFβ signaling in these processes.

The differentiation of adipose-derived stem cells (ADSCs) into tendon cells is a key process in tissue engineering and regenerative medicine. The Wnt signaling pathway plays a key role in regulating cell fate and tissue-regeneration decisions, making it a promising target for improving tendon differentiation. Photobiomodulation (PBM) is a non-invasive therapeutic approach that has been shown to modulate cellular processes, including stem cell differentiation. The aim of this review is to provide an understanding of the effects of PBM and Wnt signaling on ADSC differentiation. The complexities of interactions between PBM and dynamic Wnt pathway exist in different ways during the differentiation of ADSCs into tendon cells. The results highlight the potential therapeutic application of PBM in promoting tendon healing and regeneration. This review explores the clinical importance of PBM-mediated Wnt signaling regulation in tendon injuries. The results of this review will provide valuable information for the rational design of therapeutic strategies to enhance tendon differentiation and improve clinical outcomes and will also contribute to increasing knowledge of the synergistic relationship between PBMs, Wnt signaling pathways, and stem cell differentiation. Graphical Abstract

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.