Multi-omics analysis of human tendon adhesion reveals that ACKR1-regulated macrophage migration is involved in regeneration.

Tendon adhesion is a common complication after tendon injury with the development of accumulated fibrotic tissues without effective anti-fibrotic therapies, resulting in severe disability. Macrophages are widely recognized as a fibrotic trigger during peritendinous adhesion formation. However, different clusters of macrophages have various functions and receive multiple regulation, which are both still unknown. In our current study, multi-omics analysis including single-cell RNA sequencing and proteomics was performed on both human and mouse tendon adhesion tissue at different stages after tendon injury. The transcriptomes of over 74 000 human single cells were profiled. As results, we found that SPP1+ macrophages, RGCC+ endothelial cells, ACKR1+ endothelial cells and ADAM12+ fibroblasts participated in tendon adhesion formation. Interestingly, despite specific fibrotic clusters in tendon adhesion, FOLR2+ macrophages were identified as an antifibrotic cluster by in vitro experiments using human cells. Furthermore, ACKR1 was verified to regulate FOLR2+ macrophages migration at the injured peritendinous site by transplantation of bone marrow from Lysm-Cre;R26RtdTomato mice to lethally irradiated Ackr1-/- mice (Ackr1-/- chimeras; deficient in ACKR1) and control mice (WT chimeras). Compared with WT chimeras, the decline of FOLR2+ macrophages was also observed, indicating that ACKR1 was specifically involved in FOLR2+ macrophages migration. Taken together, our study not only characterized the fibrosis microenvironment landscape of tendon adhesion by multi-omics analysis, but also uncovered a novel antifibrotic cluster of macrophages and their origin. These results provide potential therapeutic targets against human tendon adhesion.

The subacromial bursa modulates tendon healing after rotator cuff injury in rats.

Rotator cuff injuries result in more than 500,000 surgeries annually in the United States, many of which fail. These surgeries typically involve repair of the injured tendon and removal of the subacromial bursa, a synovial-like tissue that sits between the rotator cuff and the acromion. The subacromial bursa has been implicated in rotator cuff pathogenesis and healing. Using proteomic profiling of bursa samples from nine patients with rotator cuff injury, we show that the bursa responds to injury in the underlying tendon. In a rat model of supraspinatus tenotomy, we evaluated the bursa's effect on the injured supraspinatus tendon, the uninjured infraspinatus tendon, and the underlying humeral head. The bursa protected the intact infraspinatus tendon adjacent to the injured supraspinatus tendon by maintaining its mechanical properties and protected the underlying humeral head by maintaining bone morphometry. The bursa promoted an inflammatory response in injured rat tendon, initiating expression of genes associated with wound healing, including Cox2 and Il6. These results were confirmed in rat bursa organ cultures. To evaluate the potential of the bursa as a therapeutic target, polymer microspheres loaded with dexamethasone were delivered to the intact bursae of rats after tenotomy. Dexamethasone released from the bursa reduced Il1b expression in injured rat supraspinatus tendon, suggesting that the bursa could be used for drug delivery to reduce inflammation in the healing tendon. Our findings indicate that the subacromial bursa contributes to healing in underlying tissues of the shoulder joint, suggesting that its removal during rotator cuff surgery should be reconsidered.

Bioactive fiber-reinforced hydrogel to tailor cell microenvironment for structural and functional regeneration of myotendinous junction.

Myotendinous junction (MTJ) injuries are prevalent in clinical practice, yet the treatment approaches are limited to surgical suturing and conservative therapy, exhibiting a high recurrence rate. Current research on MTJ tissue engineering is scarce and lacks in vivo evaluation of repair efficacy. Here, we developed a three-dimensional-printed bioactive fiber-reinforced hydrogel containing mesenchymal stem cells (MSCs) and Klotho for structural and functional MTJ regeneration. In a rat MTJ defect model, the bioactive fiber-reinforced hydrogel promoted the structural restoration of muscle, tendon, and muscle-tendon interface and enhanced the functional recovery of injured MTJ. In vivo proteomics and in vitro cell cultures elucidated the regenerative mechanisms of the bioactive fiber-reinforced hydrogel by modulating oxidative stress and inflammation, thus engineering an optimized microenvironment to support the survival and differentiation of transplanted MSCs and maintain the functional phenotype of resident cells within MTJ tissues, including tendon/muscle cells and macrophages. This strategy provides a promising treatment for MTJ injuries.

Curcumin Improves Functional Recovery of Ruptured Tendon by Promoting Tenogenesis via PI3K/Akt Signaling.

OBJECTIVE: In our previous study, we found that local release of curcumin from nanomicelles prevents peritendinous adhesion during Achilles tendon healing. The aim of this study is to further investigate the signaling integrated by curcumin to direct the tenogenetic program of tendon stem cells contributing to tendon healing. METHODS: A surgical model of tendon rupture and repair (TRR) was established in rats. Peritendinous adhesion and inflammation, biomechanical function, and expression of ß-catenin and epithelial cellular adhesion molecule (EpCAM) were determined. A dataset was analyzed to investigate differentially expressed genes and enriched genes related to the signaling pathways. Tendon stem cells were treated with curcumin to investigate the cellular and molecular events as well as the signaling pathway. RESULTS: In rat TRR model, curcumin treatment resulted in not only significantly decreased peritendinous inflammatory but also improved tendon functional recovery along with significantly increased expressions of EpCAM and ß-catenin. Analysis of the dataset indicated that the enriched genes were positively related to differentiation pathways but negatively related to proliferation pathways. In rat tendon stem cells, curcumin treatment inhibited proliferation but promoted differentiation. Curcumin's antioxidative activity was associated with tenogenesis. The upregulated expression of tendon lineage-specific markers was dependent on phosphatidylinositol 3'-kinase/Akt (PI3K/Akt) pathway which could be a potential mechanism of tenogenesis of curcumin treatment. CONCLUSION: Curcumin could improve tendon functional recovery via promoting tenogenesis in addition to its antioxidant and anti-inflammatory activities. Curcumin induced differentiation of tendon stem/progenitor cell into tenocytes via PI3K/Akt signaling pathway. This finding provided evidence for the application of curcumin to prevent adhesion during tendon repair.

Collagen V haploinsufficiency in female murine patellar tendons results in altered matrix engagement and cellular density, demonstrating decreased healing.

Collagen V (Col5) is a quantitatively minor component of collagen fibrils comprising tendon, however, plays a crucial role in regulation of development and dynamic healing processes. Clinically, patients with COL5a1 haploinsufficiency, known as classic Ehlers-Danlos Syndrome (cEDS), present with hyperextensible skin, joint instability and laxity, with females more likely to be affected. Previous studies in Col5-deficient mice indicated that reduced Col5a1 expression leads to a reduction in stiffness, fibril deposition, and altered fibril structure. Additionally, Col5-deficient male tendons demonstrated altered healing compared to wild-type tendons, however female mice have not yet been studied utilizing this model. Along with clinical differences between sexes in cEDS patient populations, differences in hormone physiology may be a factor influencing tendon health. Therefore, the objective of this study was to utilize a Col5a1+/ - female mouse model, to determine the effect of Col5 on tendon cell morphology, cell density, tissue composition, and mechanical properties throughout healing. We hypothesized that reduction in Col5 expression would result in an abnormal wound matrix post-injury, resulting in reduced mechanical properties compared to normal tendons. Following patellar tendon surgery, mice were euthanized at 1, 3, and 6-week post-injury. Col5-deficient tendons demonstrated altered and decreased healing compared to WT tendons. The lack of resolution in cellularity by 6-week post-injury in Col5-deficient tendons influenced the decreased mechanical properties. Stiffness did not increase post-injury in Col5-deficient mice, and collagen fiber realignment was delayed during mechanical loading. Therefore, increased Col5a1 expression post-injury is necessary to re-establish matrix engagement and cellularity throughout tendon healing.

Total flavonoids of Rhizoma drynariae improves tendon-bone healing for anterior cruciate ligament reconstruction in mice and promotes the osteogenic differentiation of bone mesenchymal stem cells by the ERR1/2-Gga1-TGF-ß/MAPK pathway.

BACKGROUND: Total flavonoids of Rhizoma drynariae (TFRD) is broadly used in the treatment of orthopedic diseases. Nevertheless, the effects and underlying mechanism of TFRD on tendon-bone healing after anterior cruciate ligament reconstruction (ACLR) remain unclear. METHODS: The ACLR mouse model was established. Hematoxylin and Eosin (HE) staining was used for histological analysis of tendon-bone healing. Western blot was utilized to detect the levels of osteogenic related factors (ALP, OCN, RUNX2). The viability and alkaline phosphatase (ALP) activity of bone mesenchymal stem cells (BMSCs) were determined by Cell Counting Kit-8 (CCK-8) and ALP assays. The interaction of estrogen related receptor alpha (ESRRA), estrogen related receptor beta (ESRRB), and golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (Gga1) was detected by luciferase reporter assays. The levels of important proteins on the TGF-ß/MAPK pathway were measured by western blot. RESULTS: TFRD improved tendon-bone healing, restored biomechanics of ACLR mice and activated the TGF-ß/MAPK pathway. TFRD treatment also enhanced the viability and osteogenic differentiation of BMSCs in vitro. Then, we demonstrated that TFRD targeted ESRRA and ESRRB to transcriptionally activate Gga1 expression. Knockdown of ESRRA, ESRRB, or Gga1 suppressed the viability and osteogenic differentiation of TFRD-induced BMSCs, which was revealed to be restored by Gga1 overexpression. The overexpression of ESRRA, ESRRB, or Gga1 was demonstrated to promote the BMSC viability and osteogenic differentiation. TGF-ß1 treatment can reverse the impact of Gga1 inhibition on osteogenic differentiation in TFRD-induced BMSCs. CONCLUSION: TFRD improves tendon-bone healing in ACLR mouse models and facilitates the osteogenic differentiation of BMSCs through the ERR1/2-Gga1-TGF-ß/MAPK pathway, which might deepen our understanding of the underlying mechanism of TFRD in tendon-bone healing.

TRIM54 alleviates inflammation and apoptosis by stabilizing YOD1 in rat tendon-derived stem cells.

Tendinopathy is a disorder of musculoskeletal system that primarily affects athletes and the elderly. Current treatment options are generally comprised of various exercise and loading programs, therapeutic modalities, and surgical interventions and are limited to pain management. This study is to understand the role of TRIM54 (tripartite motif containing 54) in tendonitis through in vitro modeling with tendon-derived stem cells (TDSCs) and in vivo using rat tendon injury model. Initially, we observed that TRIM54 overexpression in TDSCs model increased stemness and decreased apoptosis. Additionally, it rescued cells from tumor necrosis factor α-induced inflammation, migration, and tenogenic differentiation. Further, through immunoprecipitation studies, we identified that TRIM54 regulates inflammation in TDSCs by binding to and ubiquitinating YOD1. Further, overexpression of TRIM54 improved the histopathological score of tendon injury as well as the failure load, stiffness, and young modulus in vivo. These results indicated that TRIM54 played a critical role in reducing the effects of tendon injury. Consequently, these results shed light on potential therapeutic alternatives for treating tendinopathy.

TrkA-mediated sensory innervation of injured mouse tendon supports tendon sheath progenitor cell expansion and tendon repair.

Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor-ß (TGFß) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFß signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFß signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFß signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.

[Research on Runx2 gene induced differentiation of human amniotic mesenchymal stem cells into ligament fibroblasts in vitro and promotion of tendon-bone healing in rabbits].

Objective: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. Methods: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. Results: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. Conclusion: Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.

Interleukin-6 upregulates extracellular matrix gene expression and transforming growth factor ß1 activity of tendon progenitor cells.

BACKGROUND: Prolonged inflammation during tendon healing and poor intrinsic healing capacity of tendon are causal factors associated with tendon structural and functional degeneration. Tendon cells, consisting of mature tenocytes and tendon progenitor cells (TPC) function to maintain tendon structure via extracellular matrix (ECM) synthesis. Tendon cells can succumb to tissue cytokine/chemokine alterations during healing and consequently contribute to tendon degeneration. Interleukin-(IL-)1ß, IL-6 and TNFα are key cytokines upregulated in injured tendons; the specific effects of IL-6 on flexor tendon-derived TPC have not been discerned. METHODS: Passage 3 equine superficial digital flexor tendon (SDFT)-derived TPC were isolated from 6 horses. IL-6 impact on the viability (MMT assay with 0, 1, 5 and 10 ng/mL concentrations), migration (scratch motility assay at 0, 10ng/mL concentration) of TPC in monolayer culture were assessed. IL-6 effect on tendon ECM and chondrogenic gene expression (qRT-PCR), TGFß1 gene expression and activity (ELISA), and MMP-1, -3 and - 13 gene expression of TPC was evaluated. RESULTS: IL-6 decreased TPC viability and migration. IL-6 treatment at 10 ng/mL significantly up-regulated TGFß1 gene expression (6.3-fold; p = 0.01) in TPC, and significantly increased the TGFß1 concentration in cell culture supernates. IL-6 (at 10 ng/mL) significantly up-regulated both tendon ECM (COL1A1:5.3-fold, COL3A1:5.4-fold, COMP 5.5-fold) and chondrogenic (COL2A1:3.9-fold, ACAN:6.2-fold, SOX9:4.8-fold) mRNA expression in TPC. Addition of SB431542, a TGFß1 receptor inhibitor, to TPC in the presence of IL-6, attenuated the up-regulated tendon ECM and chondrogenic genes. CONCLUSION: IL-6 alters TPC phenotype during in vitro monolayer culture. Pro- and anti-inflammatory roles of IL-6 have been implicated on tendon healing. Our findings demonstrate that IL-6 induces TGFß1 activity in TPC and affects the basal TPC phenotype (as evidenced via increased tendon ECM and chondrogenic gene expressions). Further investigation of this biological link may serve as a foundation for therapeutic strategies that modulate IL-6 to enhance tendon healing.

Current progress in growth factors and extracellular vesicles in tendon healing.

Tendon injury healing is a complex process that involves the participation of a significant number of molecules and cells, including growth factors molecules in a key role. Numerous studies have demonstrated the function of growth factors in tendon healing, and the recent emergence of EV has also provided a new visual field for promoting tendon healing. This review examines the tendon structure, growth, and development, as well as the physiological process of its healing after injury. The review assesses the role of six substances in tendon healing: insulin-like growth factor-I (IGF-I), transforming growth factor ß (TGFß), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and EV. Different growth factors are active at various stages of healing and exhibit separate physiological activities. IGF-1 is expressed immediately after injury and stimulates the mitosis of various cells while suppressing the response to inflammation. VEGF, which is also active immediately after injury, accelerates local metabolism by promoting vascular network formation and positively impacts the activities of other growth factors. However, VEGF's protracted action could be harmful to tendon healing. PDGF, the earliest discovered cytokine to influence tendon healing, has a powerful cell chemotaxis and promotes cell proliferation, but it can equally accelerate the response to inflammation and relieve local adhesions. Also useful for relieving tendon adhesion is TGF- ß, which is active almost during the entire phase of tendon healing. As a powerful active substance, in addition to its participation in the field of cardiovascular and cerebrovascular vessels, tumour and chronic wounds, TGF- ß reportedly plays a role in promoting cell proliferation, activating growth factors, and inhibiting inflammatory response during tendon healing.

The potential mechanism of hypoxia-induced tenogenic differentiation of mesenchymal stem cell for tendon regeneration.

Tendon injuries account up to 50% of all musculoskeletal problems and remains a challenge to treat owing to the poor intrinsic reparative ability of tendon tissues. The natural course of tendon healing is very slow and often leads to fibrosis and disorganized tissues with inferior biomechanical properties. Mesenchymal stem cells (MSC) therapy is a promising alternative strategy to augment tendon repair due to its proliferative and multilineage differentiation potential. Hypoxic conditioning of MSC have been shown to enhance their tenogenic differentiation capacity. However, the mechanistic pathway by which this is achieved is yet to be fully defined. A key factor involved in this pathway is hypoxia-inducible factor-1-alpha (HIF-1α). This review aims to discuss the principal mechanism underlying the enhancement of MSC tenogenic differentiation by hypoxic conditioning, particularly the central role of HIF-1α in mediating activation of tenogenic pathways in the MSC. We focus on the interaction between HIF-1α with fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta 1 (TGF-ß1) in regulating MSC tenogenic differentiation pathways in hypoxic conditions. Strategies to promote stabilization of HIF-1α either through direct manipulation of oxygen tension or the use of hypoxia mimicking agents are therefore beneficial in increasing the efficacy of MSC therapy for tendon repair.

Effect of CCR2 Knockout on Tendon Biomechanical Properties in a Mouse Model of Delayed Rotator Cuff Repair.

BACKGROUND: The high incidence of incomplete or failed healing after rotator cuff repair (RCR) has led to an increased focus on the biologic factors that affect tendon-to-bone healing. Inflammation plays a critical role in the initial tendon-healing response. C-C chemokine receptor type 2 (CCR2) is a chemokine receptor linked to the recruitment of monocytes in early inflammatory stages and is associated with an increase in pro-inflammatory macrophages. The purpose of this study was to evaluate the role of CCR2 in tendon healing following RCR in C57BL/6J wildtype (WT) and CCR2-/- knockout (CCR2KO) mice in a delayed RCR model. METHODS: Fifty-two 12-week-old, male mice were allocated to 2 groups (WT and CCR2KO). All mice underwent unilateral supraspinatus tendon (SST) detachment at the initial surgical procedure, followed by a delayed repair 2 weeks later. The primary outcome measure was biomechanical testing. Secondary measures included histology, gene expression analysis, flow cytometry, and gait analysis. RESULTS: The mean load-to-failure was 1.64 ± 0.41 N in the WT group and 2.50 ± 0.42 N in the CCR2KO group (p = 0.030). The mean stiffness was 1.43 ± 0.66 N/mm in the WT group and 3.00 ± 0.95 N/mm in the CCR2KO group (p = 0.008). Transcriptional profiling demonstrated 7 differentially expressed genes (DEGs) when comparing the CCR2KO and WT groups (p < 0.05) and significant differences in Type-I and Type-II interferon pathway scores (p < 0.01). Flow cytometry demonstrated significant differences between groups for the percentage of macrophages present (8.1% for the WT group compared with 5.8% for the CCR2KO group; p = 0.035). Gait analysis demonstrated no significant differences between groups. CONCLUSIONS: CCR2KO may potentially improve tendon biomechanical properties by decreasing macrophage infiltration and/or by suppressing inflammatory mediator pathways in the setting of delayed RCR. CLINICAL RELEVANCE: CCR2 may be a promising target for novel therapeutics that aim to decrease failure rates following RCR.

Examining the Effects of In Vitro Co-Culture of Equine Adipose-Derived Mesenchymal Stem Cells With Tendon Proper and Peritenon Cells.

Tendinopathies remain the leading contributor to career-ending injuries in horses because of the complexity of tendon repair. As such, cell-based therapies like injections of adipose-derived mesenchymal stem cells (ADMSCs, or MSCs) into injured tendons are becoming increasingly popular though their long-term efficacy on a molecular and wholistic level remains contentious. Thus, we co-cultured equine MSCs with intrinsic (tendon proper) and extrinsic (peritenon) tendon cell populations to examine interactions between these cells. Gene expression for common tenogenic, perivascular, and differentiation markers was quantified at 48 and 120 hours. Additionally, cellular metabolism of proliferation was examined every 24 hours for peritenon and tendon proper cells co-cultured with MSCs. MSCs co-cultured with tendon proper or peritenon cells had altered expression profiles demonstrating trend toward tenogenic phenotype with the exception of decreases in type I collagen (COL1A1). Peritenon cells co-cultured with MSCs had a trending and significant decrease in biglycan (BGN) and CSPG4 at 48 hours and 120 hours but overall significant increases in lysyl oxidase (LOX), mohawk (MKX), and scleraxis (SCX) within 48 hours. Tendon proper cells co-cultured with MSCs also exhibited increases in LOX and SCX at 48 hours. Furthermore, cell proliferation improved overall for tendon proper cells co-cultured with MSCs. The co-culture study results suggest that adipose-derived MSCs contribute beneficially to tenogenic stimulation of peritenon or tendon proper cells.

Therapeutic potential and mechanisms of mesenchymal stem cell-derived exosomes as bioactive materials in tendon-bone healing.

Tendon-bone insertion (TBI) injuries, such as anterior cruciate ligament injury and rotator cuff injury, are the most common soft tissue injuries. In most situations, surgical tendon/ligament reconstruction is necessary for treating such injuries. However, a significant number of cases failed because healing of the enthesis occurs through scar tissue formation rather than the regeneration of transitional tissue. In recent years, the therapeutic potential of mesenchymal stem cells (MSCs) has been well documented in animal and clinical studies, such as chronic paraplegia, non-ischemic heart failure, and osteoarthritis of the knee. MSCs are multipotent stem cells, which have self-renewability and the ability to differentiate into a wide variety of cells such as chondrocytes, osteoblasts, and adipocytes. Numerous studies have suggested that MSCs could promote angiogenesis and cell proliferation, reduce inflammation, and produce a large number of bioactive molecules involved in the repair. These effects are likely mediated by the paracrine mechanisms of MSCs, particularly through the release of exosomes. Exosomes, nano-sized extracellular vesicles (EVs) with a lipid bilayer and a membrane structure, are naturally released by various cell types. They play an essential role in intercellular communication by transferring bioactive lipids, proteins, and nucleic acids, such as mRNAs and miRNAs, between cells to influence the physiological and pathological processes of recipient cells. Exosomes have been shown to facilitate tissue repair and regeneration. Herein, we discuss the prospective applications of MSC-derived exosomes in TBI injuries. We also review the roles of MSC-EVs and the underlying mechanisms of their effects on promoting tendon-bone healing. At last, we discuss the present challenges and future research directions.

Immortalized murine tenocyte cells: a novel and innovative tool for tendon research.

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 p16Ink4a/Arf and p14ARF (p19ARF 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.

Mohawk impedes angiofibrosis by preventing the differentiation of tendon stem/progenitor cells into myofibroblasts.

Adult tendons heal via fibrovascular scarring with inferior biomechanical properties. Mohawk (Mkx) emerged as a pivotal actor in tenolineage commitment. However, its precise function in tendinopathy remains poorly understood. This study investigates the cellular and molecular mechanisms underlying Mkx' role in fibrovascular healing. Human samples were collected to test fibrovascular markers. We then performed RNAseq on Mkx-/- mice compared to their wild type littermates to decipher Mkx regulome. We therefore sought to reproduce TSPCs transition to myofibroblasts in-vitro by over-expressing MyoD and followed by phenotypic and experimental cells' characterization using microscopy, qRT-PCR, flow cytometry sorting, presto-blue cell viability assay and immunofluorescence. Two different in vivo models were used to assess the effect of the MyoD-expressing myofibroblasts: transplantation in the dorsal area of immunodeficient mice and in an adult Achilles tendon injury model. To prevent angiofibrosis, we tested the molecule Xav939 and proceeded with histological stainings, q-RT PCR transcriptional quantification of angifibrotic markers, mechanical tests, and immunofluorescence. Tendinopathy samples showed fibrovascular healing with decreased tenolineage phenotype. Transcriptomic analysis of Mkx-/- tendons revealed myofibroblast-associated biological processes. Over-expression of MyoD in WT tendon stem progenitor cells (TSPCs) gave rise to myofibroblasts reprogramming in-vitro and fibrovascular scarring in-vivo. MKX directly binds to MyoD promoter and underlies global regulative processes related to angiogenesis and Wnt signaling pathway. Blocking Wnt signaling with the small molecule Xav393 resulted in higher histological and biomechanical properties. Taken together, our data provide the first in vivo and in-vitro evidence of tendon stem progenitor cells to myofibroblasts transition and show improved tendon healing via angiofibrosis modulation, thus opening potential therapeutic avenues to treat tendinopathy patients.

TGF-ß2 enhances expression of equine bone marrow-derived mesenchymal stem cell paracrine factors with known associations to tendon healing.

BACKGROUND: Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix proteins that contribute to their ability to support tissue healing and regeneration. Both the transcriptome and the secretome of MSCs can be altered by treating the cells with cytokines, but neither have been thoroughly investigated following treatment with the specific cytokine transforming growth factor (TGF)-ß2. METHODS: RNA-sequencing and western blotting were used to compare gene and protein expression between untreated and TGF-ß2-treated equine bone marrow-derived MSCs (BM-MSCs). A co-culture system was utilized to compare equine tenocyte migration during co-culture with untreated and TGF-ß2-treated BM-MSCs. RESULTS: TGF-ß2 treatment significantly upregulated gene expression of collagens, extracellular matrix molecules, and growth factors. Protein expression of collagen type I and tenascin-C was also confirmed to be upregulated in TGF-ß2-treated BM-MSCs compared to untreated BM-MSCs. Both untreated and TGF-ß2-treated BM-MSCs increased tenocyte migration in vitro. CONCLUSIONS: Treating equine BM-MSCs with TGF-ß2 significantly increases production of paracrine factors and extracellular matrix molecules important for tendon healing and promotes the migration of tenocytes in vitro.

Development of a continuum damage model to predict accumulation of sub-failure damage in tendons.

Many painful and physically debilitating conditions involve sub-failure mechanical damage to seemingly intact connective tissues such as tendons and ligaments. We found that the amount of denatured collagen in rat tail tendon (RTT) fascicles increased over experiments of cyclic loading to a constant load level (creep cyclic fatigue) with fluorescently tagged collagen hybridizing peptides (CHPs) that bind to denatured collagen. To better understand tendon sub-failure damage progression, computational modeling of tendon materials via finite element analysis in FEBio has been conducted. The objective of this project was to develop, implement, and test the ability of a new continuum damage mechanics (CDM) model in FEBio to represent the sub-failure damage behavior seen in our RTT fascicle creep cyclic fatigue experimental data. There appeared to be two distinct mechanisms responsible for the creep cyclic fatigue softening behavior of RTT fascicles over the number of cycles to failure: the preconditioning effect and overall collagen damage. In our finite element (FE) models, the RTT fascicle undamaged elastic constitutive material was composed of a matrix and fibers described by the Coupled Veronda-Westmann and exponential-linear materials. This undamaged elastic material was convolved with a modified CDM model adapted from Balzani et al., in 2012. The novelty of the Balzani damage model is the inclusion of two interrelated mechanisms described as continuous and discontinuous damage. The continuous damage formulation calculates damage accumulation during the loading and reloading of each new cycle, while the discontinuous damage approach accumulates damage from the maximum strain over the loading history to the current time. We modified the Balzani damage model formulations to represent exponential and sigmoidal increases in damage marked by the preconditioning effect and collagen damage in RTT fascicles as functions of continuous and discontinuous damage. The original Balzani damage model was first verified, then the modified CDM model was implemented into FEBio and used to reproduce the sample specific experimental creep cyclic fatigue stress-strain data as well as predict incremental cyclic fatigue. The resulting model will be useful for future experimental and computational studies of damage mechanics to understand tendon pathologies.

VEGFA-Enriched Exosomes from Tendon-Derived Stem Cells Facilitate Tenocyte Differentiation, Migration, and Transition to a Fibroblastic Phenotype.

Tendon-derived stem cells (TDSCs) play a vital role in repair of rotator cuff tear injuries by secreting paracrine proteins that regulate resident cell functions. Secreted exosomes may play a role in tendon injury repair by mediating intercellular communication; however, the detailed mechanisms by which TDSC-derived exosomes affect tenocyte development remain unknown. Here, we examined the effects of exosomes isolated from conditioned medium of TDSCs on tenocyte differentiation, migration, and transition to a fibroblastic phenotype in vitro. Successful isolation of exosomes from TDSCs was confirmed by high expression levels of CD81, CD63, CD9, and TSG101. Treatment with TDSC-derived exosomes promoted the growth and migration of cultured rat tenocytes, and increased the levels of the fibrosis markers collagen I, collagen III, scleraxis, tenascin C, and α-smooth muscle actin. Furthermore, vascular endothelial growth factor A (VEGFA) expression was higher in TDSC-derived exosomes than in TDSCs, and genetic knockdown of VEGFA suppressed the stimulatory effect of TDSC-derived exosomes on tenocyte development. Overall, these results demonstrate that VEGFA-enriched exosomes isolated from TDSCs promote differentiation and migration of cultured tenocytes and their transition to a fibroblastic phenotype. These data provide a new potential clinical treatment strategy for tendon injury.