Tendon Markers Research Articles

Electrohydrodynamic-Printed Dual-Triphase Microfibrous Scaffolds Reshaping the Lipidomic Profile for Enthesis Healing in a Rat Rotator Cuff Repair Model.

Rotator cuff injuries often result in chronic pain and functional limitations due to retears and scar formation at the enthesis. This study assess the efficacy of electrohydrodynamic-printed microfibrous dual-triphase scaffolds (DTSs), designed to optimize enthesis repair. These scaffolds, composed of polycaprolactone enhanced with nanohydroxyapatite, nano-magnesium-oxide, and kartogenin demonstrate significant biological advantages. In vitro, the scaffolds support over 95% stem cell viability and promote enhanced expression of critical markers such as tenomodulin (TNMD), sex-determining region Y-Box transcription factor 9 (SOX-9), and runt-related transcription factor 2 (RUNX-2). Enhanced expressions of tendon markers tenomodulinand scleraxis (SCX) are noted, alongside significant upregulation of chondrocyte and osteoblast markers. In vivo, these scaffolds significantly improve the biomechanical properties of the repaired enthesis, with a maximum failure load of 27.0 ±4.2 N and ultimate stress of 5.5 ±1.0MPa at 6 weeks postimplantation. Lipidomic analysis indicates substantial regulation of phospholipids such as phosphatidylcholine and phosphatidylserine, highlighting the scaffold's capacity to modulate biochemical pathways critical for tissue repair and regeneration. This study underscores the potential of DTS to improve clinical outcomes in rotator cuff injury treatment by enhancing cellular differentiation, biomechanical properties, and biochemical environment, setting a foundation for personalized treatment strategies in tendon-bone repair.

The histone H3K9 methyltransferase G9a regulates tendon formation during development

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 Sox9Cre/+ mice with G9afl/fl mice to generate G9afl/fl; Sox9Cre/+ 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.

Galangin Promotes Tendon Repair Mediated by Tendon-Derived Stem Cells through Activating the TGF-β1/Smad3 Signaling Pathway.

Tendon injury is a prevalent orthopedic disease that currently lacks effective treatment. Galangin (GLN) is a vital flavonoid found abundantly in galangal and is known for its natural activity. This study aimed to investigate the GLN-mediated molecular mechanism of tendon-derived stem cells (TDSCs) in tendon repair. The TDSCs were characterized using alkaline phosphatase staining, alizarin red S staining, oil red O staining, and flow cytometry. The effect of GLN treatment on collagen deposition was evaluated using Sirius red staining and quantitative (q)PCR, while a Western bot was used to assess protein levels and analyze pathways. Results showed that GLN treatment not only increased the collagen deposition but also elevated the mRNA expression and protein levels of multiple tendon markers like collagen type I alpha 1 (COL1A1), decorin (DCN) and tenomodulin (TNMD) in TDSCs. Moreover, GLN was also found to upregulate the protein levels of transforming growth factor β1 (TGF-β1) and p-Smad3 to activate the TGF-β1/Smad3 signaling pathway, while GLN mediated collagen deposition in TDSCs was reversed by LY3200882, a TGF-β receptor inhibitor. The study concluded that GLN-mediated TDSCs enhanced tendon repair by activating the TGF-β1/Smad3 signaling pathway, suggesting a novel therapeutic option in treating tendon repair.

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension.

Today's challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

Recombinant lysyl oxidase effects on embryonic tendon cell phenotype and behavior.

Lysyl oxidase (LOX) plays an important role in the elaboration of tendon mechanical properties during embryonic development by mediating enzymatic collagen crosslinking. We previously showed recombinant LOX (rLOX) treatment of developing tendon significantly increased LOX-mediated collagen crosslink density to enhance tendon mechanical properties at different stages of tissue formation. Working toward the future development of rLOX-based therapeutic strategies to enhance mechanical properties of tendons that are compromised, such as after injury or due to abnormal development, this study characterized the direct effects of rLOX treatment on embryonic tendon cells from different stages of tissue formation. Tendon cell morphology, proliferation rate, proliferative capacity, and metabolic activity were not affected by rLOX treatment. Tenogenic phenotype was stable with rLOX treatment, reflected by no change in cell morphology or tendon marker messenger RNA (mRNA) levels assessed by reverse-transcription polymerase chain reaction. Collagen mRNA levels also remained constant. Matrix metalloproteinase-9 expression levels were downregulated in later stage tendon cells, but not in earlier stage cells, whereas enzyme activity levels were undetected. Bone morphogenetic protein-1 (BMP-1) expression was upregulated in earlier stage tendon cells, but not in later stage cells. Furthermore, BMP-1 activity was unchanged when intracellular LOX enzyme activity levels were upregulated in both stage cells, suggesting exogenous rLOX may have entered the cells. Based on our data, rLOX treatment had minimal effects on tendon cell phenotype and behaviors. These findings will inform future development of LOX-focused treatments to enhance tendon mechanical properties without adverse effects on tendon cell phenotype and behaviors.

Pro-inflammatory activity of long noncoding RNA FOXD2-AS1 in Achilles tendinopathy

Achilles tendinopathy is a prevalent clinical problem that plagues athletes and general populations. Achilles tendon healing is a complex process, and so far, there is no successful long-term solution to Achilles tendinopathy in the field of microsurgery due to its poor natural regeneration ability. Limitations in understanding the pathogenesis of Achilles tendon development and Achilles tendon injury hinder clinical treatment developments. There is an increasing demand for innovative conservative treatments that can improve Achilles tendon injury. In this study, a Sprague–Dawley rat model of Achilles tendinopathy was established. Lentiviral vectors that interfere with the expression of FOXD2-AS1, miR-21-3p, or PTEN were injected every 3 days. Rats were euthanized after 3 weeks, and the effect of FOXD2-AS1, miR-21-3p, or PTEN on Achilles tendon healing was analyzed by histological observation, biomechanical test, and examinations of inflammatory factors and tendon markers. As measured, downregulating FOXD2-AS1 or upregulating miR-21-3p improved histological structure, suppressed inflammation, promoted the expression of tendon markers, and optimized the biomechanical properties of Achilles tendon. Upregulating PTEN was capable of reversing the promoting effect of inhibition of FOXD2-AS1 on Achilles tendon healing. As concluded, deficiency of FOXD2-AS1 accelerates the healing of Achilles tendon injury and improves tendon degeneration by regulating the miR-21-3p/PTEN axis and promoting the activation of the PI3K/AKT signaling pathway.

THE POTENTIAL OF MAGNETIC CELL SHEET PATCHES IN TENDON REGENERATION

Tissue engineering and regenerative medicine (TERM) hold the promise to provide therapies for injured tendons despite the challenging cues of tendon niche and the lack of specific factors to guide regeneration. The emerging potential of magnetic responsiveness and magnetic nanoparticles (MNPs) functionalities offers new perspectives to tackle TERM challenges. Moreover, pulsed electromagnetic field (PEMF) is FDA approved for orthopaedics with potential to control inflammation upon injury. We previously demonstrated that magnetic cell-sheets assisted by PEMF trigger the inflammation resolution by modulating cytokine-enriched environments [1]. To further understand the potential of magnetically assisted living patches, we have recently conducted in vivo studies using a rat patellar defect model.After labeling of human adipose stem cells with iron oxide MNPs for 16h, magCSs were cultured up to 3 days in α-MEM medium under non-magnetic or PEMF conditions. MagCSs were evaluated by immunocytochemistry, and real time RT-PCR for tendon markers. Cell metabolic activity was also assessed by MTS and ECM proteins quantified by Sirius Red/Fast Green.The MagCSs effect in ameliorating healing was assessed after implantation in window defects created in the patellar tendon of rats. PEMF was externally applied (3mT, 70Hz) 3d/week for 1h (magnetotherapy). After 4 and 8w, tendons were histologically characterized for immune-detection of tendon and inflammatory markers, and for Perls van Gieson and HE stains. Blood and detoxification organs were screened for inflammatory mediators and biodistribution of MNPs, respectively.In vitro results suggest that PEMF stimulates cellular metabolic activity, influences protein synthesis and the deposition of collagen and non-collagenous proteins is significantly increased compared to non-magnetic conditions. No adverse reactions, as infection or swelling, were observed after surgery or during follow-up. After 8w, magCSs remained at the implantation site and no MNPs were detected on detoxification organs. Plasma levels of IL1α, β, IL6 and TNFα assessed by multiplex assay were below detectable values (<12.5pg/ml).Thus, the combination of cell sheets and magnetic technologies hold promise for the development of living tendon substitutes.Acknowledgement to ERC-COG MagTendon772817, H2020 Achilles 810850, FCT - 2020.01157.CEECIND.

IPSC-derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles tendon regeneration.

Tendons and ligaments have a poor innate healing capacity, yet account for 50% of musculoskeletal injuries in the United States. Full structure and function restoration postinjury remains an unmet clinical need. This study aimed to assess the application of novel three dimensional(3D) printed scaffolds and induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) overexpressing the transcription factor Scleraxis (SCX, iMSCSCX+ ) as a new strategy for tendon defect repair. The polycaprolactone (PCL) scaffolds were fabricated by extrusion through a patterned nozzle or conventional round nozzle. Scaffolds were seeded with iMSCSCX+ and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSCSCX+ -seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence.iMSCSCX+ -seeded on microgrooved scaffolds showed upregulation of tendon markers and increased organization and linearity of cells compared to non-patterned scaffolds in vitro. In vivo gait analysis showed improvement in the Scaffold + iMSCSCX+ -treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold + iMSCSCX+ group compared with the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold + iMSCSCX+ group. This study demonstrates the potential of 3D-printed scaffolds with cell-instructive surface topography seeded with iMSCSCX+ as an approach to tendon defect repair. Further studies of cell-scaffold constructs can potentially revolutionize tendon reconstruction by advancing the application of 3D printing-based technologies toward patient-specific therapies that improve healing and functional outcomes at both the cellular and tissue level.

Fullerenol inhibits tendinopathy by alleviating inflammation.

Tendinopathy is a common disease in orthopaedics, seriously affecting tendon functions. However, the effects of non-surgical treatment on tendinopathy are not satisfactory and surgical treatments possibly impair the function of tendons. Biomaterial fullerenol has been proved to show good anti-inflammatory effects on various inflammatory diseases. For in vitro experiments, primary rat tendon cells (TCs) were treated by interleukin-1 beta (IL-1β) combined with aqueous fullerenol (5, 1, 0.3μg/mL). Then inflammatory factors, tendon-related markers, migration and signaling pathways were detected. For in vivo experiments, rat tendinopathy model was constructed by local injection of collagenase into Achilles tendons of rats and fullerenol (0.5, 1mg/mL) was locally injected 7days after collagenase injection. Inflammatory factors and tendon-related markers were also investigated. Fullerenol with good water-solubility showed excellent biocompatibility with TCs. Fullerenol could increase expression of tendon-related factors (Collagen I and tenascin C) and decrease expression of inflammatory factors (matrix metalloproteinases-3, MMP-3, and MMP-13) and reactive oxygen species (ROS) level. Simultaneously, fullerenol slowed the migration of TCs and inhibited activation of Mitogen-activated protein kinase (MAPK) signaling pathway. Fullerenol also attenuated tendinopathy in vivo, including reduction of fiber disorders, decrease of inflammatory factors and increase of tendon markers. In summary, fullerenol is a promising biomaterial that can be used to treat tendinopathy.

Micropattern Silk Fibroin Film Facilitates Tendon Repair In Vivo and Promotes Tenogenic Differentiation of Tendon Stem/Progenitor Cells through the α2β1/FAK/PI3K/AKT Signaling Pathway In Vitro.

Tendon injuries are common clinical disorders. Due to the limited regeneration ability of tendons, tissue engineering technology is often used as an adjuvant treatment. This study explored the molecular pathways underlying micropattern SF film-regulated TSPC propensity and their repairing effects to highlight the application value of micropattern SF films. First, we characterized the physical properties of the micropattern SF films and explored their repairing effects on the injured tendons in vivo. Then, we seeded TSPCs on SF films in vitro and determined the micropattern SF film-induced gene expression and activation of signaling pathways in TSPCs through high-throughput RNA sequencing and proteomics assays. The results of in vivo studies suggested that micropattern SF films can promote remodeling of the injured tendon. In addition, immunohistochemistry (IHC) results showed that tendon marker genes were significantly increased in the micropattern SF film repair group. Transcriptomic and proteomic analyses demonstrated that micropattern SF film-induced genes and proteins in TSPCs were mainly enriched in the focal adhesion kinase (FAK)/actin and phosphoinositide 3-kinase (PI3K)/AKT pathways. Western blot analysis showed that the expression of integrins α2β1, tenascin-C (TNC), and tenomodulin (TNMD) and the phosphorylation of AKT were significantly increased in the micropattern SF film group, which could be abrogated by applying PI3K/AKT inhibitors. Micropattern SF films modified by water annealing can promote remodeling of the injured tendon in vivo and regulate the tendon differentiation of TSPCs through the α2β1/FAK/PI3K/AKT signaling pathway in vitro. Therefore, they have great medical value in tendon repair.

Directing iPSC differentiation into iTenocytes using combined scleraxis overexpression and cyclic loading.

Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of iPSC-derived mesenchymal stromal-like cells (iMSCs). Scxis the single direct molecular regulator of tendon differentiation known to date. Bone marrow-derived (BM-)MSCs were used as reference. Scx overexpression alone resulted in significantly higher upregulation of tenogenic markers in iMSCs compared to BM-MSCs. Mechanoregulation is known to be a central element guiding tendon development and healing. Mechanical stimulation combined with Scx overexpression resulted in morphometric and cytoskeleton-related changes, upregulation of early and late tendon markers, and increased extracellular matrix deposition and alignment, and tenomodulin perinuclear localization in iMSCs. Our findings suggest that these cells can be differentiated into tenocytes and might be a better candidate for tendon cell therapy applications than BM-MSCs.

Novel roles of tendon in the postnatal growth and expansion of TMJ condyle

The Temporomandibular joint (TMJ) is one of the most complex joints in the human body. TMJ is composed of the temporal bone, a disc and a movable mandibular condyle with abundant tendon attachments. Tendon has been thought to play the sole function of transmitting muscle forces to stabilize joints, yet it is largely unclear why tendon undergoes ectopic ossification in trauma or diseases, and whether it has any direct contribution to skeletal formation. This study aimed to investigate the full biological significance of tendon in TMJ growth. We first discovered that the TMJ condyle is composed of a well‐established cartilage head and an overlooked “bony head” that grows after birth and continuously expands throughout the lifespan with little signs of remodeling. Mouse X‐ray images (Fig.1a) showed little change in the cartilage head’s volume but a continuous expansion in the bony head’s mass with a low mineral content from 1 to 5 months (Fig.1b). Toluidine blue staining showed TMJ condyle had a large area of tendon attachment extending down to ramus (Fig.1c, white dotted line in lower magnification), defined by regions of tendon, interface, and TFB (Fig.1c1). The TFB morphology was distinct from endosteum‐formed bone (EFB, Fig.1c1), cartilage‐formed bone (CFB, Fig.1c2, rich in cartilage residual), or periosteum‐formed bone (PFB, Fig.1c3) in cell shape and distribution, and ECM. TEM images further revealed that the osteocytes in the TFB were large in size, irregular in shape, had small nuclei but numerous ERs and Golgi complexes, and were embedded in ECM rich in fibropositors. In contrast, the osteocytes in EFB, CFB or PFB were spindle‐shaped with larger nuclei but fewer ERs and Golgi complexes (Fig.1d). To reveal the cell source of the bony head, cell lineage tracing were used. Tracing data showed that most CFB cells originate from Col10a1+ hypertrophic chondrocytes, whereas the interface and TFB were derived from Scx+ cells (Fig.1e). RNAscope displayed high levels of Thbs4 (Thrombospondin‐4, a tendon marker) and SOST (a potent inhibitor of Wnt signaling secreted by osteocytes) mRNA in TFB at bony head (Fig.1f). The Scx‐CreERT2 tracing combined with IHC staining showed TFB maintained a mixed ECM of bone (Col1), cartilage (Aggrecan) and tendon (Periostin, Fig.1g). To further determine the role of tendon lineage in condyle expansion, we generated Scx‐CreERT2; R26RDTA (carrying a loxP‐flanked stop cassette associated with an attenuated diphtheria toxin fragment A, DTA, for the ablation of cells when Cre is active). Deletion of Scx+ cells greatly reduced the size of bony head (Fig.1h) and the thickness of interface with few Scx+/Col1+ bone cells in P28 DTA mice (Fig.1i); In conclusion, our study tendon cells, beyond their conventional role in joint movement, are key players for the postnatal growth and expansion of TMJ condyle (Fig.1j).

Coordinated patterning of zebrafish caudal fin symmetry by a central and two peripheral organizers.

Caudal fin symmetry characterizes teleosts and likely contributes to their evolutionary success. However, the coordinated development and patterning of skeletal elements establishing external symmetry remains incompletely understood. We explore the spatiotemporal emergence of caudal skeletal elements in zebrafish to consider evolutionary and developmental origins of caudal fin symmetry. Transgenic reporters and skeletal staining reveal that the hypural diastema-defining gap between hypurals 2 and 3 forms early and separates progenitors of two plates of connective tissue. Two sets of central principal rays (CPRs) synchronously, sequentially, and symmetrically emerge around the diastema. The two dorsal- and ventral-most rays (peripheral principal rays, PPRs) arise independently and earlier than adjacent CPRs. Muscle and tendon markers reveal that different muscles attach to CPR and PPR sets. We propose that caudal fin symmetry originates from a central organizer that establishes the hypural diastema and bidirectionally patterns surrounding tissue into two plates of connective tissue and two mirrored sets of CPRs. Further, two peripheral organizers unidirectionally specify PPRs, forming a symmetric "composite" fin derived from three fields. Distinct CPR and PPR ontogenies may represent developmental modules conferring ray identities, muscle connections, and biomechanical properties. Our model contextualizes mechanistic studies of teleost fin morphological variation.

Promethazine Downregulates Wnt/β-Catenin Signaling and Increases the Biomechanical Forces of the Injured Achilles Tendon in the Early Stage of Healing

Background: Wnt/β-catenin signaling suppresses the differentiation of cultured tenocytes, but its roles in tendon repair remain mostly elusive. No chemical compounds are currently available to treat tendon injury. Hypothesis: We hypothesized that the inhibition of Wnt/β-catenin signaling would accelerate tendon healing. Study Design: Controlled laboratory study. Methods: Tendon-derived cells (TDCs) were isolated from rat Achilles tendons. The right Achilles tendon was injured via a dermal punch, while the left tendon was sham operated. A Wnt/β-catenin inhibitor, IWR-1, and an antihistamine agent, promethazine (PH), were locally and intramuscularly injected, respectively, for 2 weeks after surgery. The healing tendons were histologically and biomechanically evaluated. Results: The amount of β-catenin protein was increased in the injured tendons from postoperative weeks 0.5 to 2. Inhibition of Wnt/β-catenin signaling by IWR-1 in healing tendons improved the histological abnormalities and decreased β-catenin, but it compromised the biomechanical properties. As we previously reported that antihistamine agents suppressed Wnt/β-catenin signaling in human chondrosarcoma cells, we examined the effects of antihistamines on TDCs. We found that a first-generation antihistamine agent, PH, increased the expression of the tendon marker genes Mkx and Tnmd in TDCs. Intramuscular injection of PH did not improve histological abnormalities, but it decreased β-catenin in healing tendons and increased the peak force and stiffness of the healing tendons on postoperative week 2. On postoperative week 8, however, the biomechanical properties of vehicle-treated tendons became similar to those of PH-treated tendons. Conclusion: IWR-1 and PH suppressed Wnt/β-catenin signaling and improved the histological abnormalities of healing tendons. IWR-1, however, compromised the biomechanical properties of healing tendons, whereas PH improved them. Clinical Relevance: PH is a candidate repositioned drug that potentially accelerates tendon repair.

Axin2-lineage cells contribute to neonatal tendon regeneration

ABSTRACT Purpose Tendon injuries are a challenging clinical problem with few treatment options. Identifying the molecular regulators of tendon is required for the development of new therapies. While the Wnt pathway is critical for the maintenance and differentiation of many tissues, the role of Wnt signaling in tendon cell biology remains largely unexplored. Methods The effects of Wnt activation were tested in vitro using neonatal tendon-derived cells cultured in 2D and 3D conditions. The inducible Axin2CreERT2 was then used to label Axin2+ cells in vivo and cells were traced during neonatal tendon regeneration. Results We showed that activation of Wnt signaling results in proliferation of neonatal tendon cells. While tendon marker expression was inhibited by Wnt activation under 2D conditions, Scx expression was not affected under 3D uniaxial tension, suggesting that the microenvironment contextualizes tendon cell response to Wnt signaling. Using an in vivo model of neonatal tendon regeneration, we further showed that Wnt signaling cells comprise a subpopulation of tenocyte and epitenon cells that proliferate after injury and are recruited during regeneration. Discussion Collectively, these studies suggest that Wnt signaling may play a role in tendon cell proliferation, differentiation, and regeneration.

Nonwoven-based gelatin/polycaprolactone membrane loaded with ERK inhibitor U0126 for treatment of tendon defects

BackgroundTendon is a major component of musculoskeletal system connecting the muscles to the bone. Tendon injuries are very common orthopedics problems leading to impeded motion. Up to now, there still lacks effective treatments for tendon diseases.MethodsTendon stem/progenitor cells (TSPCs) were isolated from the patellar tendons of SD rats. The expression levels of genes were evaluated by quantitative RT-PCR. Immunohistochemistry staining was performed to confirm the presence of tendon markers in tendon tissues. Bioinformatics analysis of data acquired by RNA-seq was used to find out the differentially expressed genes. Rat patellar tendon injury model was used to evaluate the effect of U0126 on tendon injury healing. Biomechanical testing was applied to evaluate the mechanical properties of newly formed tendon tissues.ResultsIn this study, we have shown that ERK inhibitor U0126 rather PD98059 could effectively increase the expression of tendon-related genes and promote the tenogenesis of TSPCs in vitro. To explore the underlying mechanisms, RNA sequencing was performed to identify the molecular difference between U0126-treated and control TSPCs. The result showed that GDF6 was significantly increased by U0126, which is an important factor of the TGFβ superfamily regulating tendon development and tenogenesis. In addition, NBM (nonwoven-based gelatin/polycaprolactone membrane) which mimics the native microenvironment of the tendon tissue was used as an acellular scaffold to carry U0126. The results demonstrated that when NBM was used in combination with U0126, tendon healing was significantly promoted with better histological staining outcomes and mechanical properties.ConclusionTaken together, we have found U0126 promoted tenogenesis in TSPCs through activating GDF6, and NBM loaded with U0126 significantly promoted tendon defect healing, which provides a new treatment for tendon injury.

Nonsurgical treatment reduces tendon inflammation and elevates tendon markers in early healing.

Operative treatment is assumed to provide superior outcomes to nonoperative (conservative) treatment following Achilles tendon rupture, however, this remains controversial. This study explores the effect of surgical repair on Achilles tendon healing. Rat Achilles tendons (n = 101) were bluntly transected and were randomized into groups receiving repair or non-repair treatments. By 1 week after injury, repaired tendons had inferior mechanical properties, which continued to 3- and 6-weekpost-injury, evidenced by decreased dynamic modulus and failure stress. Transcriptomics analysis revealed >7000 differentially expressed genes between repaired and non-repaired tendons after 1-week post-injury. While repaired tendons showed enriched inflammatory gene signatures, non-repaired tendons showed increased tenogenic, myogenic, and mechanosensitive gene signatures, with >200-fold enrichment in Tnmd expression. Analysis of gastrocnemius muscle revealed elevated MMP activity in tendons receiving repair treatment, despite no differences in muscle fiber morphology. Transcriptional regulation analysis highlighted that the highest expressed transcription factors in repaired tendons were associated with inflammation (Nfκb, SpI1, RelA, andStat1), whereas non-repaired tendons expressed markers associated with tissue development and mechano-activation (Smarca1, Bnc2, Znf521, Fbn1, andGli3). Taken together, these data highlight distinct differences in healing mechanism occurring immediately following injury and provide insights for new therapies to further augment tendons receiving repaired and non-repaired treatments.

Discovery of Muscle-Tendon Progenitor Subpopulation in Human Myotendinous Junction at Single-Cell Resolution.

The myotendinous junction (MTJ) is a complex and special anatomical area that connects muscles and tendons, and it is also the key to repairing tendons. Nevertheless, the anatomical structure and connection structure of MTJ, the cluster and distribution of cells, and which cells are involved in repairing the tissue are still unclear. Here, we analyzed the cell subtype distribution and function of human MTJ at single-cell level. We identified four main subtypes, including stem cell, muscle, tendon, and muscle-tendon progenitor cells (MTP). The MTP subpopulation, which remains the characteristics of stem cells and also expresses muscle and tendon marker genes simultaneously, may have the potential for bidirectional differentiation. We also found the muscle-tendon progenitor cells were distributed in the shape of a transparent goblet; muscle cells first connect to the MTP and then to the tendon. And after being transplanted in the MTJ injury model, MTP exhibited strong regenerative capability. Finally, we also demonstrated the importance of mTOR signaling for MTP maintenance by in vitro addition of rapamycin and in vivo validation using mTOR-ko mice. Our research conducted a comprehensive analysis of the heterogeneity of myotendinous junction, discovered a special cluster called MTP, provided new insights into the biological significance of myotendinous junction, and laid the foundation for future research on myotendinous junction regeneration and restoration.

Cell autonomous TGFβ signaling is essential for stem/progenitor cell recruitment into degenerative tendons.

SummaryUnderstanding cell recruitment in damaged tendons is critical for improvements in regenerative therapy. We recently reported that targeted disruption of transforming growth factor beta (TGFβ) type II receptor in the tendon cell lineage (Tgfbr2ScxCre) resulted in resident tenocyte dedifferentiation and tendon deterioration in postnatal stages. Here we extend the analysis and identify direct recruitment of stem/progenitor cells into the degenerative mutant tendons. Cre-mediated lineage tracing indicates that these cells are not derived from tendon-ensheathing tissues or from a Scleraxis-expressing lineage, and they turned on tendon markers only upon entering the mutant tendons. Through immunohistochemistry and inducible gene deletion, we further find that the recruited cells originated from a Sox9-expressing lineage and their recruitment was dependent on cell autonomous TGFβ signaling. The cells identified in this study thus differ from previous reports of cell recruitment into injured tendons and suggest a critical role for TGFβ signaling in cell recruitment, providing insights that may support improvements in tendon repair.

Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-β, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-β3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-β3-induced tenogenic differentiation. The MSC were incubated with and without TGF-β3 (10 ng/ml), Y-27632 (10 μM), or both. On day 1 and day 3, the signalling pathway of TGF-β and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-β3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-β3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-β3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-β3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.