Assessing Bioprinted Functionalized Grafts for Biological Tendon Augmentation In Vitro.

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

Transcriptomics reveals dynamic changes in the "gene profiles" of rat supraspinatus tendon at three different time points after diabetes induction.

OBJECTIVE: There is increasing evidence that type 2 diabetes mellitus (T2DM) is an independent risk factor for the occur of tendinopathy. Therefore, this study is the first to explore the dynamic changes of the "gene profile" of supraspinatus tendon in rats at different time points after T2DM induction through transcriptomics, providing potential molecular markers for exploring the pathogenesis of diabetic tendinopathy. METHODS: A total of 40 Sprague-Dawley rats were randomly divided into normal (NG, n = 10) and T2DM groups (T2DM, n = 30) and subdivided into three groups according to the duration of diabetes: T2DM-4w, T2DM-8w, and T2DM-12w groups; the duration was calculated from the time point of T2DM rat model establishment. The three comparison groups were set up in this study, T2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG. Differentially expressed genes (DEGs) in 3 comparison groups were screened. The intersection of the three comparison groups' DEGs was defined as key genes that changed consistently in the supraspinatus tendon after diabetes induction. Cluster analysis, gene ontology (GO) functional annotation analysis and Kyoto encyclopedia of genes and genomes (KEGG) functional annotation and enrichment analysis were performed for DEGs. RESULTS: T2DM-4w group vs. NG, T2DM-8w group vs. NG, and T2DM-12w group vs. NG detected 519 (251 up-regulated and 268 down-regulated), 459 (342 up-regulated and 117 down-regulated) and 328 (255 up-regulated and 73 down-regulated) DEGs, respectively. 103 key genes of sustained changes in the supraspinatus tendon following induction of diabetes, which are the first identified biomarkers of the supraspinatus tendon as it progresses through the course of diabetes.The GO analysis results showed that the most significant enrichment in biological processes was calcium ion transmembrane import into cytosol (3 DEGs). The most significant enrichment in cellular component was extracellular matrix (9 DEGs). The most significant enrichment in molecular function was glutamate-gated calcium ion channel activity (3 DEGs). The results of KEGG pathway enrichment analysis showed that there were 17 major pathways (p < 0.05) that diabetes affected supratinusculus tendinopathy, including cAMP signaling pathway and Calcium signaling pathway. CONCLUSIONS: Transcriptomics reveals dynamic changes in the"gene profiles"of rat supraspinatus tendon at three different time points after diabetes induction. The 103 DEGs identified in this study may provide potential molecular markers for exploring the pathogenesis of diabetic tendinopathy, and the 17 major pathways enriched in KEGG may provide new ideas for exploring the pathogenesis of diabetic tendinopathy.

Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

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.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

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-ß (TGFß) 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 TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 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. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 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.

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.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Collagen VI Deficiency Impairs Tendon Fibroblasts Mechanoresponse in Ullrich Congenital Muscular Dystrophy.

The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons.

Activation of CGRP receptor-mediated signaling promotes tendon-bone healing.

Calcitonin gene-related peptide (CGRP), an osteopromotive neurotransmitter with a short half-life, shows increase while calcitonin receptor-like (CALCRL) level is decreased at the early stage in bone fractures. Therefore, the activation of CALCRL-mediated signaling may be more critical to promote the tendon-bone healing. We found CGRP enhanced osteogenic differentiation of BMSCs through PKA/CREB/JUNB pathway, contributing to improved sonic hedgehog (SHH) expression, which was verified at the tendon-bone interface (TBI) in the mice with Calcrl overexpression. The osteoblast-derived SHH and slit guidance ligand 3 were reported to favor nerve regeneration and type H (CD31hiEMCNhi) vessel formation, respectively. Encouragingly, the activation or inactivation of CALCRL-mediated signaling significantly increased or decreased intensity of type H vessel and nerve fiber at the TBI, respectively. Simultaneously, improved gait characteristics and biomechanical performance were observed in the Calcrl overexpression group. Together, the gene therapy targeting CGRP receptor may be a therapeutic strategy in sports medicine.

FGF-stimulated tendon cells embrace a chondrogenic fate with BMP7 in newt tissue culture.

Newts can regenerate functional elbow joints after amputation at the joint level. Previous studies have suggested the potential contribution of cells from residual tendon tissues to joint cartilage regeneration. A serum-free tissue culture system for tendons was established to explore cell dynamics during joint regeneration. Culturing isolated tendons in this system, stimulated by regeneration-related factors, such as fibroblast growth factor (FGF) and platelet-derived growth factor, led to robust cell migration and proliferation. Moreover, cells proliferating in an FGF-rich environment differentiated into Sox9-positive chondrocytes upon BMP7 introduction. These findings suggest that FGF-stimulated cells from tendons may aid in joint cartilage regeneration during functional elbow joint regeneration in newts.

In vitro collagen biomarkers in mechanically stimulated human tendon cells: a systematic review.

OBJECTIVE: The aim of this study was to comprehensively examine and summarize the available in vitro evidence regarding the relationship between mechanical stimulation and biomarkers of collagen synthesis in human-derived tendon cells. METHODS: Systematic review with narrative analyses and risk of bias assessment guided by the Health Assessment and Translation tool. The electronic databases MEDLINE (Ovid), EMBASE (Ovid), CENTRAL (Ovid) and COMPENDEX (Engineering Village) were systematically searched from inception to 3 August 2023. Inclusion criteria encompassed English language, original experimental, or quasi-experimental in vitro publications that subjected human tendon cells to mechanical stimulation, with collagen synthesis (total collagen, type I, III, V, XI, XII, and XIV) and related biomarkers (matrix metalloproteinases, transforming growth factor ß, scleraxis, basic fibroblast growth factor) as outcomes. RESULTS: Twenty-one publications were included. A pervasive definite high risk of bias was evident in all included studies. Owing to incomplete outcome reporting and heterogeneity in mechanical stimulation protocols, planned meta-analyses were unfeasible. Reviewed data suggested that human tendon cells respond to mechanical stimulation with increased synthesis of collagen (e.g., COL1A1, procollagen, total soluble collagen, etc.), scleraxis and several matrix metalloproteinases. Results also indicate that mechanical stimulation dose magnitude may influence synthesis in several biomarkers. CONCLUSIONS: A limited number of studies, unfortunately characterized by a definite high risk of bias, suggest that in vitro mechanical stimulation primarily increases type I collagen synthesis by human tendon cells. Findings from this systematic review provide researchers and clinicians with biological evidence concerning the possible beneficial influence of exercise and loading on cellular-level tendon adaptation.

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.

HPF1 regulates tendon stem/progenitor cell senescence and tendon repair via PARP1-mediated poly-ADP ribosylation of HuR.

BACKGROUND: Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear. OBJECTIVE: This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs. METHODS: Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated ß-galactosidase (SA-ß-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD. RESULTS: Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence. CONCLUSIONS: HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.

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.

Examining the Potential of Vitamin C Supplementation in Tissue-Engineered Equine Superficial Digital Flexor Tendon Constructs.

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

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.