RESUMEN
Inflammation, corticosteroids, and loading all affect tendon healing, with an interaction between them. However, underlying mechanisms behind the effect of corticosteroids and the interaction with loading remain unclear. The aim of this study was to investigate the role of dexamethasone during tendon healing, including specific effects on tendon cells. Rats (n = 36) were randomized to heavy loading or mild loading, the Achilles tendon was transected, and animals were treated with dexamethasone or saline. Gene and protein analyses of the healing tendon were performed for extracellular matrix-, inflammation-, and tendon cell markers. We further tested specific effects of dexamethasone on tendon cells in vitro. Dexamethasone increased mRNA levels of S100A4 and decreased levels of ACTA2/α-SMA, irrespective of load level. Heavy loading + dexamethasone reduced mRNA levels of FN1 and TenC (p < 0.05), while resolution-related genes were unaltered (p > 0.05). In contrast, mild loading + dexamethasone increased mRNA levels of resolution-related genes ANXA1, MRC1, PDPN, and PTGES (p < 0.03). Altered protein levels were confirmed in tendons with mild loading. Dexamethasone treatment in vitro prevented tendon construct formation, increased mRNA levels of S100A4 and decreased levels of SCX and collagens. Dexamethasone during tendon healing appears to act through immunomodulation by promoting resolution, but also through an effect on tendon cells.
Asunto(s)
Tendón Calcáneo , Dexametasona , Traumatismos de los Tendones , Cicatrización de Heridas , Dexametasona/farmacología , Animales , Ratas , Cicatrización de Heridas/efectos de los fármacos , Traumatismos de los Tendones/tratamiento farmacológico , Traumatismos de los Tendones/metabolismo , Tendón Calcáneo/efectos de los fármacos , Tendón Calcáneo/metabolismo , Tendón Calcáneo/lesiones , Tendón Calcáneo/patología , Proteína de Unión al Calcio S100A4/metabolismo , Proteína de Unión al Calcio S100A4/genética , Masculino , Anexina A1/metabolismo , Anexina A1/genética , Actinas/metabolismo , Actinas/genética , Colágeno/metabolismo , Ratas Sprague-Dawley , Tendones/efectos de los fármacos , Tendones/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice BásicoRESUMEN
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 ß1 (TGF-ß1) and p-Smad3 to activate the TGF-ß1/Smad3 signaling pathway, while GLN mediated collagen deposition in TDSCs was reversed by LY3200882, a TGF-ß receptor inhibitor. The study concluded that GLN-mediated TDSCs enhanced tendon repair by activating the TGF-ß1/Smad3 signaling pathway, suggesting a novel therapeutic option in treating tendon repair.
Asunto(s)
Flavonoides , Transducción de Señal , Proteína smad3 , Células Madre , Tendones , Factor de Crecimiento Transformador beta1 , Flavonoides/farmacología , Flavonoides/química , Factor de Crecimiento Transformador beta1/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Proteína smad3/metabolismo , Proteína smad3/antagonistas & inhibidores , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Madre/citología , Tendones/citología , Tendones/metabolismo , Tendones/efectos de los fármacos , Ratas , Células Cultivadas , Ratas Sprague-Dawley , Traumatismos de los Tendones/tratamiento farmacológico , Traumatismos de los Tendones/metabolismoRESUMEN
BACKGROUND: Tendons are important dense fibrous structures connecting muscle to bone, and tendon stem cells (TDSCs) affect their repair and regeneration. The role of TDSC-derived exosomes (TDSC-Exos) is still being unexplored; therefore, this study aimed to investigate the protective effect of TDSC-Exos on tenocytes. METHODS: The TDSCs and tenocytes were all derived from Sprague Dawley (SD) rats. The expression of positive and negative markers of TDSCs were detected by flow cytometry, and the multi-differentiation ability was also detected to identify TDSCs. Exos were derived from TDSCs using ultracentrifugation; furthermore, Exos enriched with microRNA(miR)-377-3p were generated from TDSCs stably overexpressing miR-377-3p after transfection, identified with transmission electron microscopy (TEM), western blot and PKH26 staining assay. Moreover, the cell functions of tenocytes were evaluated by MTT, EdU, transwell, and flow cytometry. Dual luciferase reporter and RNA pull-down assays were used to verify the binding sites of miR-337-3p and caspase3 (CASP3) predicted by Targetscan. RESULTS: Exos (miR-337-3p) were taken up by tenocytes, and promoted the proliferation, migration, and invasion and suppressed the apoptosis of tenocytes in a dose-dependent manner. Bioinformatics analysis showed that CASP3 was a target of miR-377-3p, which was further verified by luciferase and RNA pull-down assays. Moreover, over-expressed CASP3 reversed the effects of Exos (miR-337-3p) on cell functions of tenocytes. CONCLUSIONS: Our findings suggest that Exos derived from miR-337-3p over-expressing TDSCs could potentially protect against tenocyte apoptosis by regulating CASP3. This novel therapeutic approach holds promise for the treatment of tendon injury, offering a glimmer of hope for improved patient outcomes.
Asunto(s)
Apoptosis , Caspasa 3 , Exosomas , MicroARNs , Ratas Sprague-Dawley , Células Madre , Tendones , Tenocitos , Animales , MicroARNs/genética , MicroARNs/metabolismo , Exosomas/metabolismo , Exosomas/genética , Apoptosis/fisiología , Ratas , Caspasa 3/metabolismo , Caspasa 3/genética , Tenocitos/metabolismo , Células Madre/metabolismo , Tendones/metabolismo , Tendones/citología , Proliferación Celular/fisiología , Células Cultivadas , Masculino , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/patología , Movimiento CelularRESUMEN
Tendon injuries typically display limited reparative capacity, often resulting in suboptimal outcomes and an elevated risk of recurrence or rupture. While cytokines of the IL-6 family are primarily recognised for their inflammatory properties, they also have multifaceted roles in tissue regeneration and repair. Despite this, studies examining the association between IL-6 family cytokines and tendon repair remained scarce. gp130, a type of glycoprotein, functions as a co-receptor for all cytokines in the IL-6 family. Its role is to assist in the transmission of signals following the binding of ligands to receptors. RCGD423 is a gp130 modulator. Phosphorylation of residue Y759 of gp130 recruits SHP2 and SOCS3 and inhibits activation of the STAT3 pathway. In our study, RCGD423 stimulated the formation of homologous dimers of gp130 and the phosphorylation of Y759 residues without the involvement of IL-6 and IL-6R. Subsequently, the phosphorylated residues recruited SHP2, activating the downstream ERK and AKT pathways. These mechanisms ultimately promoted the migration ability of tenocytes and matrix synthesis, especially collagen I. Moreover, RCGD423 also demonstrated significant improvements in collagen content, alignment of collagen fibres, and biological and biomechanical function in a rat Achilles tendon injury model. In summary, we demonstrated a promising gp130 modulator (RCGD423) that could potentially enhance tendon injury repair by redirecting downstream signalling of IL-6, suggesting its potential therapeutic application for tendon injuries.
Asunto(s)
Tendón Calcáneo , Movimiento Celular , Receptor gp130 de Citocinas , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Proto-Oncogénicas c-akt , Ratas Sprague-Dawley , Tenocitos , Animales , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptor gp130 de Citocinas/metabolismo , Tendón Calcáneo/metabolismo , Tendón Calcáneo/lesiones , Tendón Calcáneo/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Ratas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tenocitos/metabolismo , Tenocitos/efectos de los fármacos , Tenocitos/fisiología , Colágeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Masculino , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/tratamiento farmacológicoRESUMEN
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.
Asunto(s)
Movimiento Celular , Macrófagos , Regeneración , Humanos , Animales , Macrófagos/metabolismo , Ratones , Tendones/metabolismo , Tendones/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Traumatismos de los Tendones/patología , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/genética , Proteómica , Femenino , MultiómicaRESUMEN
Heterotopic ossification (HO), the pathological formation of bone within soft tissues such as tendon and muscle, is a notable complication resulting from severe injury. While soft tissue injury is necessary for HO development, the specific molecular pathology responsible for trauma-induced HO remains a mystery. The previous study detected abnormal autophagy function in the early stages of tendon HO. Nevertheless, it remains to be determined whether autophagy governs the process of HO generation. Here, trauma-induced tendon HO model is used to investigate the relationship between autophagy and tendon calcification. In the early stages of tenotomy, it is observed that autophagic flux is significantly impaired and that blocking autophagic flux promoted the development of more rampant calcification. Moreover, Gt(ROSA)26sor transgenic mouse model experiments disclosed lysosomal acid dysfunction as chief reason behind impaired autophagic flux. Stimulating V-ATPase activity reinstated both lysosomal acid functioning and autophagic flux, thereby reversing tendon HO. This present study demonstrates that autophagy-lysosomal dysfunction triggers HO in the stages of tendon injury, with potential therapeutic targeting implications for HO.
Asunto(s)
Autofagia , Modelos Animales de Enfermedad , Lisosomas , Ratones Transgénicos , Osificación Heterotópica , Tendones , Osificación Heterotópica/metabolismo , Osificación Heterotópica/genética , Osificación Heterotópica/patología , Animales , Autofagia/fisiología , Ratones , Lisosomas/metabolismo , Tendones/metabolismo , Tendones/patología , Tendones/fisiopatología , Tenotomía/métodos , Masculino , Traumatismos de los Tendones/fisiopatología , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/patología , Ratones Endogámicos C57BLRESUMEN
Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.
Asunto(s)
Células Endoteliales , Traumatismos de los Tendones , Animales , Ratones , Humanos , Células Endoteliales/metabolismo , Estudios Longitudinales , Tendones/fisiología , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/cirugía , Colágeno/metabolismo , Ingeniería de Tejidos/métodosRESUMEN
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.
Asunto(s)
Osificación Heterotópica , Complejo de la Endopetidasa Proteasomal , Proteína Homóloga de Ras Enriquecida en el Cerebro , Transducción de Señal , Ubiquitina , Animales , Ratas , Complejo de la Endopetidasa Proteasomal/metabolismo , Osificación Heterotópica/metabolismo , Osificación Heterotópica/etiología , Osificación Heterotópica/patología , Transducción de Señal/efectos de los fármacos , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Ubiquitina/metabolismo , Masculino , Osteogénesis/efectos de los fármacos , Tendones/metabolismo , Tendones/patología , Ratas Sprague-Dawley , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/patología , Traumatismos de los Tendones/complicaciones , Proteolisis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Tendón Calcáneo/metabolismo , Tendón Calcáneo/patología , Tendón Calcáneo/lesiones , Modelos Animales de Enfermedad , Ubiquitinación , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células Madre/metabolismo , Células Madre/efectos de los fármacosRESUMEN
Abstract Purpose: To evaluate the effect of transforming growth factor β1 (TGF-β1) on tendon-to-bone reconstruction of rotator cuff tears. Methods: Seventy-two rat supraspinatus tendons were transected and reconstructed in situ. At 8 and 16 weeks, specimens of three groups; that is control, L-dose (low dose), and H-dose (high dose) were harvested and underwent a biomechanical test to evaluate the maximum load and stiffness values. Histology sections of the tendon-to-bone interface were identified by hematoxylin-eosin or Masson trichrome stain. Collagen type III was observed by picric acid sirius red staining under polarized light. The level of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) was measured by the enzyme-linked immunosorbent assay (ELISA) method. Results: Collagen type III of the H-dose group had a significant difference in histology structure compared with the L-dose group (P<0.05). The maximum load and stiffness decreased significantly in the control group compared with the values of the L-dose and H-dose groups. The stiffness among the three groups differed significantly at the same postoperative time (P<0.05). Interestingly, progressive reestablishment of collagen type III affected tendon-to-bone healing significantly in the later stages. Conclusion: The H-dose was associated with an increased collagen type III morphology stimulated by TGF-β1.