RESUMEN
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.
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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 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.
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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
CRISPR-Cas13 holds substantial promise for tissue repair through its RNA editing capabilities and swift catabolism. However, conventional delivery methods fall short in addressing the heightened inflammatory response orchestrated by macrophages during the acute stages of tendon injury. In this investigation, macrophage-targeting cationic polymers are systematically screened to facilitate the entry of Cas13 ribonucleic-protein complex (Cas13 RNP) into macrophages. Notably, SPP1 (OPN encoding)-producing macrophages are recognized as a profibrotic subtype that emerges during the inflammatory stage. By employing ROS-responsive release mechanisms tailored for macrophage-targeted Cas13 RNP editing systems, the overactivation of SPP1 is curbed in the face of an acute immune microenvironment. Upon encapsulating this composite membrane around the tendon injury site, the macrophage-targeted Cas13 RNP effectively curtails the emergence of injury-induced SPP1-producing macrophages in the acute phase, leading to diminished fibroblast activation and mitigated peritendinous adhesion. Consequently, this study furnishes a swift RNA editing strategy for macrophages in the inflammatory phase triggered by ROS in tendon injury, along with a pioneering macrophage-targeted carrier proficient in delivering Cas13 into macrophages efficiently.
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Sistemas CRISPR-Cas , Macrófagos , Traumatismos de los Tendones , Macrófagos/metabolismo , Animales , Ratones , Traumatismos de los Tendones/terapia , Traumatismos de los Tendones/genética , Inmunoterapia , Edición de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células RAW 264.7 , Osteopontina/genética , Osteopontina/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Both acute and chronic Achilles tendon ruptures are affected by alterations in the extracellular matrix during the healing process of the tendon. Yet, these alterations in gene expression patterns are not well characterized. PURPOSE: To characterize temporal and spatial differences in gene expression patterns after an Achilles tendon rupture and to evaluate if cells from chronic Achilles tendon ruptures have the same ability to form new tendon tissue (tendon constructs) as healthy tendon cells. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 35 patients with surgically treated Achilles tendon ruptures were included in the study and divided into 3 groups: acute (<4 weeks), short-term chronic (1-6 months), and long-term chronic (>6 months). Biopsy specimens were collected during surgical repair and were used to analyze the gene expression within the different groups and to compare mRNA levels in the proximal and distal tendon ends. A complementary in vitro experiment was performed to evaluate if cells from chronic Achilles tendon ruptures can form tendon constructs. RESULTS: The mRNA levels for COL1A1 and COL3A1 were significantly higher in the short-term chronic group compared with the acute group (P < .05). Both MMP-1 and MMP-13 had the highest mRNA levels in the acute group (P < .01) compared with the long-term chronic group, while MMP-2 had the highest mRNA level in the short-term chronic group. Significant differences between the proximal and distal tendon ends were only detected for the monocyte and macrophage marker CD163 (P < .05), which was more expressed proximally. Cells extracted from chronic Achilles tendon ruptures displayed a similar ability and effectiveness to form tendon constructs as healthy tendon cells. CONCLUSION: A high collagenase gene activity after an Achilles tendon rupture indicated possible rapid matrix degradation in the acute phase. Chronic ruptures appeared to initiate the healing process even before treatment, indicated by the higher expression of collagen in the short-term chronic group. Cells from chronic Achilles tendon ruptures also displayed an ability to form new tendon tissue in vitro. CLINICAL RELEVANCE: The study shows a rapid increase in collagenase gene expression, which could lead to matrix degradation that continues for months after an Achilles tendon rupture.
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Tendón Calcáneo , Traumatismos del Tobillo , Traumatismos de los Tendones , Humanos , Interleucina-6 , Tendón Calcáneo/cirugía , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/cirugía , Traumatismos de los Tendones/patología , Rotura/cirugía , Colagenasas , ARN Mensajero , Expresión Génica , Resultado del TratamientoRESUMEN
BACKGROUND: Due to its limited blood supply and irregular mechanical loading, the Achilles tendon is the most frequently ruptured tendon. Despite the rising incidence of acute Achilles tendon rupture (AATR), the optimal treatment remains controversial. Missed diagnoses and delayed treatments lead to poor outcomes and limited treatment options. This study aimed to identify potential biomarkers for diagnosing and developing therapies for AATR. METHODS: We employed the coupled isobaric tag for relative and absolute quantitation-liquid chromatography-electrospray ionization-tandem mass spectrometry approach to investigate protein expression in tissues from AATR patients. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to identify differentially expressed proteins (DEPs) between AATR patients and healthy individuals. A protein-protein interaction (PPI) network of DEPs was constructed using the Search Tool for the Retrieval of Interacting Genes. The screened hub genes were selectively verified by immunohistochemical staining. RESULTS: We identified 410 DEPs between AATR patients and controls. The DEPs were significantly enriched in GO terms such as the extracellular region, extracellular region part, and defense response, as well as KEGG pathways, including complement and coagulation cascades, focal adhesion, and regulation of actin cytoskeleton. The main hub nodes in the PPI network comprised fibronectin 1 (FN1), major histocompatibility complex, class I, B (HLA-B), filamin A (FLNA), heat shock 27-kDa protein 1 (HSPB1), heat shock protein family A member 5 (HSPA5), apolipoprotein A4 (APOA4), and myosin IC (MYO1C). Although APOA4 and collagens I, II, and III were detectable in healthy tendons, immunohistochemical staining confirmed higher expression of these proteins in the acutely ruptured Achilles tendon. CONCLUSIONS: Our findings lay a foundation for further molecular studies of AATR. Inflammation and age-related degeneration may contribute to the pathogenesis of AATR. Moreover, the identified DEPs could be potential biomarkers for AATR diagnosis and treatment.
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Tendón Calcáneo , Traumatismos de los Tendones , Humanos , Proteómica/métodos , Mapas de Interacción de Proteínas , Biomarcadores , Traumatismos de los Tendones/diagnóstico , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/terapiaRESUMEN
Tendon injury is one of the most common disorders of the musculoskeletal system, with a higher likelihood of occurrence in elderly individuals and athletes. In posthealing tendons, two undesirable consequences, tissue fibrosis and a reduction in mechanical properties, usually occur, resulting in an increased probability of rerupture or reinjury; thus, it is necessary to propose an appropriate treatment. Currently, most methods do not sufficiently modulate the tendon healing process and restore the function and structure of the injured tendon to those of a normal tendon, since there is still inadequate information about the effects of multiple cellular and other relevant signaling pathways on tendon healing and how the expression of their components is regulated. microRNAs are vital targets for promoting tendon repair and can modulate the expression of biological components in signaling pathways involved in various physiological and pathological responses. miRNAs are a type of noncoding ribonucleic acid essential for regulating processes such as cell proliferation, differentiation, migration and apoptosis; inflammatory responses; vascularization; fibrosis; and tissue repair. This article focuses on the biogenesis response of miRNAs while presenting their mechanisms in tendon healing with perspectives and suggestions.
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MicroARNs , Traumatismos de los Tendones , Humanos , Anciano , MicroARNs/genética , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/patología , Tendones/patología , Diferenciación Celular , FibrosisRESUMEN
OBJECTIVE: The study aimed to investigate the polymorphism of fibrillin-2 (FBN2) and elastin genes in patients with Achilles tendon rupture and to compare the results with a control group of participants who did not experience such an injury. METHODS: In this prospective study, 106 consecutive patients in whom traumatic Achilles tendon rupture was diagnosed and treated were included. The control group consisted of randomly selected 92 athletes (10 women and 82 men) 85 of whom had practiced sports in the past, aged 40-76 years, who during their sports career did not experience Achilles tendon ruptures. Material for genetic tests was obtained by the swab from the oral cavity epithelium of all the study population. RESULTS: 102 (96%) of patients with traumatic Achilles tendon ruptures were people with polymorphism B or heterozygotes for the elastin gene. 97 (92%) of patients with traumatic Achilles tendon ruptures were people with polymorphism B and heterozygotes for the FBN2 gene. Patients with homozygote A of the elastin gene and homozygote A of the FBN2 gene demonstrated a considerably lower incidence rate of sport-related Achilles tendon rupture. The type of sport that led to the Achilles tendon rupture and the amount of experience practicing it, as well as BMI and drug usage, did not contribute to a higher rate of incidence of any additional musculoskeletal problems or a longer time to return to their pre-injury sports activity. Polymorphisms of the fibrillin 2 (P=.0001) and elastin (P=.0009) genes impact the occurrence of traumatic injury to the Achilles tendon. However, it does not affect the length of full recovery time (P =.2251). CONCLUSION: Minimally invasive and, above all, safe collection of genetic material from the epithelium of the oral cavity in order to assess the polymorphic state of the FBN and elastin genes may allow the identification of a group of players at risk of Achilles tendon rupture resulting in long-term injury, which will significantly affect their sports career in the future. LEVEL OF EVIDENCE: Level II, Prognostic Study.
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Tendón Calcáneo , Traumatismos de los Tendones , Femenino , Humanos , Masculino , Tendón Calcáneo/lesiones , Elastina/genética , Fibrilina-2 , Polimorfismo Genético , Estudios Prospectivos , Rotura/genética , Rotura/cirugía , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/cirugíaRESUMEN
BACKGROUND: Tendon injuries are among the most common musculoskeletal disorders. Celecoxib possesses an effective anti-inflammatory activity in the tendon injury treatment. Lactoferrin has a great potential for the tendon regeneration. However, the efficacy of celecoxib combined with lactoferrin in the treatment of tendon injury has not been reported. In this study, we aimed to investigate the effect of celecoxib and lactoferrin on tendon injury and repair, and screen for the crucial genes associated with the tendon injury and repair. METHODS: The rat tendon injury models were established and divided into four groups: normal control group (n = 10), tendon injury model group (n = 10), celecoxib treatment group (n = 10), and celecoxib + lactoferrin treatment group (n = 10). Then, RNA sequencing was performed to identify differentially expressed lncRNAs (DElncRNAs), miRNAs (DEmiRNAs) and mRNAs (DEmRNAs) in celecoxib treatment group and celecoxib + lactoferrin treatment group. Next, autophagy/hypoxia/ferroptosis/pyroptosis-related DEmRNAs were further identified. Subsequently, functional enrichment, protein-protein interaction (PPI) network and transcriptional regulatory network construction for these genes were performed. RESULTS: The animal study demonstrated that combinational administration of celecoxib with lactoferrin rescued the harmful effects caused by celecoxib in the treatment of tendon injury. Compared to tendon injury model group, 945 DEmRNAs, 7 DEmiRNAs and 34 DElncRNAs were obtained in celecoxib treatment group, and 493 DEmRNAs, 8 DEmiRNAs and 21 DElncRNAs were obtained in celecoxib + lactoferrin treatment group, respectively. Subsequently, 376 celecoxib + lactoferrin treatment group-specific DEmRNAs were determined. Then, 25 DEmRNAs associated with autophagy/hypoxia/ferroptosis/pyroptosis were identified. CONCLUSIONS: Several genes, such as, Ppp1r15a, Ddit4, Fos, Casp3, Tgfb3, Hspb1 and Hspa8, were identified to be associated with tendon injury and repair.
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Ferroptosis , Traumatismos de los Tendones , Animales , Ratas , Celecoxib/farmacología , Lactoferrina/genética , Regulación Neoplásica de la Expresión Génica , Piroptosis , Redes Reguladoras de Genes , Traumatismos de los Tendones/tratamiento farmacológico , Traumatismos de los Tendones/genéticaRESUMEN
Tendon injury repair has been a clinical challenge, and little is known about tendon healing scar generation, repair, and regeneration mechanisms. To explore the cellular composition of tendon tissue and analyze cell populations and signaling pathways associated with tendon repair, in this paper, single-cell sequencing data was used for data mining and seven cell subsets were annotated in the tendon tissue, including fibroblasts, tenocytes, smooth muscle cells, endothelial cells, macrophages, T cells, and plasma cells. According to cell group interaction network analysis, pattern 4 composed of macrophages was an important communication pattern in tendon injury. Furthermore, the heterogeneity of M1 macrophages in tendons, the correlation of KEGG enriched pathway with inflammatory response, and the core regulatory role of the transcription factor NFKB and REL were observed; in addition, the heterogeneity of T cell isoforms in tendons was found and indicated that different isotypes of T cells involve in different roles of tendon injury and repair. This study demonstrated the heterogeneity of M1 macrophages and T cells in the tendon tissue, being involved in different physiological processes such as tendon injury and healing, providing new thinking insights and basis for subsequent clinical treatment of tendon injury.
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Traumatismos de los Tendones , Transcriptoma , Células Endoteliales/metabolismo , Humanos , Isoformas de Proteínas/metabolismo , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/metabolismo , Factores de Transcripción , Transcriptoma/genéticaRESUMEN
Platelet-rich plasma (PRP) is a widely used autologous treatment for tendon injuries in clinics. Platelets (PLTs) are a major source of high mobility group box1 (HMGB1) that is gaining attention as a chemoattractant that can recruit stem cells to the wound area to enhance healing of injured tissues; however, the contribution of PLT HMGB1 in wounded tendon healing remains unexplored. This study investigated the effect of PLT HMGB1 within PRP on tendon healing using PLT HMGB1 knockout (KO) and GFP mice. A window defect was created in the patellar tendons of both groups of mice, and wounds were treated with either saline, PRP isolated from PLT HMGB1-KO mice, or PRP isolated from GFP mice. Seven days post-treatment, animals were sacrificed and analyzed by gross inspection, histology, and immunostaining for characteristic signs of tendon healing and repair. Our results showed that in comparison to mice treated with PRP from PLT HMGB1-KO mice, wounds treated with PRP from GFP mice healed faster and exhibited a better organization in tendon structure. Mice treated with PRP from PLT HMGB1-KO mice produced tendon tissue with large premature wound areas and low cell densities. However, wounds of PLT HMGB1-KO mice showed better healing with PRP from HMGB1-KO mice compared to saline treatment. Moreover, wounds treated with PRP from GFP mice had increased extracellular HMGB1, decreased CD68, increased stem cell markers CD146 and CD73, and increased collagen III protein expression levels compared to those treated with PRP from PLT HMGB1-KO mice. Thus, PLT HMGB1 within PRP plays an important role in tendon wound healing by decreasing inflammation, increasing local HMGB1 levels, and recruiting stem cells to the wound area in the tendon. Our findings also suggest that the efficacy of PRP treatment for tendon injuries in clinics may depend on PLT HMGB1 within PRP preparations.
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Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Plasma Rico en Plaquetas/fisiología , Traumatismos de los Tendones/terapia , Cicatrización de Heridas , 5'-Nucleotidasa/metabolismo , Animales , Antígeno CD146/metabolismo , Colágeno Tipo III/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Plasma Rico en Plaquetas/metabolismo , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/metabolismo , Factores de Tiempo , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
Tendon injuries are one of the most common musculoskeletal disorders that cause considerable morbidity and significantly compromise the patients' quality of life. The innate limited regenerative capacity of tendon poses a substantial treating challenge for clinicians. MicroRNAs (miRNAs) are a family of small non-coding RNAs that play a vital role in orchestrating many biological processes through post-transcriptional regulation. Increasing evidence reveals that miRNA-based therapeutics may serve as an innovative strategy for the treatment of tendon pathologies. In this review, we briefly present miRNA biogenesis, the role of miRNAs in tendon cell biology and their involvement in tendon injuries, followed by a summary of current miRNA-based approaches in tendon tissue engineering with a special focus on attenuating post-injury fibrosis. Next, we discuss the advantages of miRNA-functionalized scaffolds in achieving sustained and localized miRNA administration to minimize off-target effects, and thus hoping to inspire the development of effective miRNA delivery platforms specifically for tendon tissue engineering. We envision that advancement in miRNA-based therapeutics will herald a new era of tendon tissue engineering and pave a way for clinical translation for the treatments of tendon disorders.
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MicroARNs , Traumatismos de los Tendones , Humanos , MicroARNs/genética , Calidad de Vida , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/terapia , Tendones , Ingeniería de TejidosRESUMEN
PURPOSE: This study aimed to screen the entire genome for genetic markers associated with risk for Achilles tendon injury. METHODS: A genome-wide association analysis was performed using data from the Kaiser Permanente Research Board and the UK Biobank. Achilles tendon injury cases were identified based on electronic health records from the Kaiser Permanente Research Board databank and the UK Biobank from individuals of European ancestry. Genome-wide association analyses from both cohorts were tested for Achilles tendon injury using a logistic regression model adjusting for sex, height, weight, and race/ethnicity using allele counts for single nucleotide polymorphisms (SNP). Previously identified genes within the literature were also tested for association with Achilles tendon injury. RESULTS: There were a total of 12,354 cases of Achilles tendon injury and 483,080 controls within the two combined cohorts, with 67 SNP in three chromosomal loci demonstrating a genome-wide significant association with Achilles tendon injury. The first locus contains a single SNP (rs183364169) near the CDCP1 and TMEM158 genes on chromosome 3. The second locus contains 65 SNP in three independently segregating sets near the MPP7 gene on chromosome 10. The last locus contains a single SNP (rs4454832) near the SOX21 and GPR180 genes on chromosome 13. The current data were used to test 14 candidate genes previously reported to show an association with Achilles tendon injury, but none showed a significant association (all P > 0.05). CONCLUSION: Three loci were identified as potential risk factors for Achilles tendon injury and deserve further validation and investigation of molecular mechanisms.
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Tendón Calcáneo/lesiones , Estudio de Asociación del Genoma Completo , Traumatismos de los Tendones/genética , Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Receptores Acoplados a Proteínas G/genética , Factores de Riesgo , Factores de Transcripción SOXB2/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: Environmental conditions strongly influence the healing capacity of connective tissues. Well-vascularized extrasynovial tendons typically undergo a robust wound-healing process following transection and repair. In contrast, avascular intrasynovial tendons do not mount an effective repair response. The current study tests the hypothesis that flexor tendons, as a function of their synovial environment, exhibit unique inflammatory, angiogenic, and metabolic responses to injury and repair. METHODS: Flexor tendons present a distinct opportunity to test the study hypothesis, as they have proximal regions that are extrasynovial and distal regions that are intrasynovial. In an internally controlled study design, the second and fifth forepaw flexor tendons were transected and repaired in either the extrasynovial or the intrasynovial anatomical region. Histological, gene expression, and proteomics analyses were performed at 3 and 7 days to define the early biological events that drive synovial environment-dependent healing responses. RESULTS: Uninjured intrasynovial tendons were avascular, contained high levels of proteoglycans, and expressed inflammatory factors, complement proteins, and glycolytic enzymes. In contrast, extrasynovial tendons were well vascularized, contained low levels of proteoglycans, and were enriched in inflammation inhibitors and oxidative phosphorylation enzymes. The response to injury and repair was markedly different between the 2 tendon regions. Extrasynovial tendons displayed a robust and rapid neovascularization response, increased expression levels of complement proteins, and an acute shift in metabolism to glycolysis, whereas intrasynovial tendons showed minimal vascularity and muted inflammatory and metabolic responses. CONCLUSIONS: The regional molecular profiles of intact and healing flexor tendons revealed extensive early differences in innate immune response, metabolism, vascularization, and expression of extracellular matrix as a function of the synovial environment. These differences reveal mechanisms through which extrasynovial tendons heal more effectively than do intrasynovial tendons. CLINICAL RELEVANCE: To improve outcomes after operative repair, future treatment strategies should promote features of extrasynovial healing, such as enhanced vascularization and modulation of the complement system and/or glucose metabolism.
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Traumatismos de los Tendones , Tendones/fisiología , Cicatrización de Heridas/fisiología , Animales , Proteínas del Sistema Complemento/análisis , Perros , Proteínas de la Matriz Extracelular/análisis , Femenino , Miembro Anterior , Perfilación de la Expresión Génica , Glucólisis , Mediadores de Inflamación/análisis , Modelos Animales , Neovascularización Fisiológica , Fosforilación Oxidativa , Proteoglicanos/análisis , Distribución Aleatoria , Membrana Sinovial , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/patología , Traumatismos de los Tendones/cirugía , Tendones/irrigación sanguínea , Tendones/metabolismo , Tendones/patología , Factores de TiempoRESUMEN
A multipotent cell population co-expressing a basic-helix-loop-helix transcription factor scleraxis (Scx) and SRY-box 9 (Sox9) has been shown to contribute to the establishment of entheses (tendon attachment sites) during mouse embryonic development. The present study aimed to investigate the involvement of Scx+/Sox9+ cells in the postnatal formation of fibrocartilaginous entheses and in the healing process after injury, using ScxGFP transgenic mice. We demonstrate that Scx+/Sox9+ cells are localized in layers at the insertion site during the postnatal formation of fibrocartilaginous entheses of supraspinatus tendon until postnatal 3 weeks. Further, these cells were rarely seen at postnatal 6 weeks, when mature fibrocartilaginous entheses were formed. Furthermore, we investigated the involvement of Scx+/Sox9+ cells in the healing process after supraspinatus tendon enthesis injury, comparing the responses of 20- and 3-week-old mice. In the healing process of 20-week-old mice with disorganized fibrovascular tissue in response to injury, a small number of Scx+/Sox9+ cells transiently appeared from 1 week after injury, but they were rarely seen at 4 weeks after injury. Meanwhile, in 3-week-old mice, a thin layer of fibrocartilaginous tissue with calcification was formed at healing enthesis at 4 weeks after injury. From 1 to 2 weeks after injury, more Scx+/Sox9+ cells, widely distributed at the injured site, were seen compared with the 20-week-old mice. At 4 weeks after injury, these cells were located near the surface of the recreated fibrocartilaginous layer. This spatiotemporal localization pattern of Scx+/Sox9+ cells at the injured enthesis in our 3-week-old mouse model was similar to that in postnatal fibrocartilaginous enthesis formation. These findings indicate that Scx+/Sox9+ cells may have a role as entheseal progenitor-like cells during postnatal maturation of fibrocartilaginous entheses and healing after injury in a manner similar to that seen in embryonic development.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factor de Transcripción SOX9/genética , Traumatismos de los Tendones/terapia , Cicatrización de Heridas/genética , Animales , Linaje de la Célula/genética , Modelos Animales de Enfermedad , Fibrocartílago/crecimiento & desarrollo , Fibrocartílago/lesiones , Fibrocartílago/metabolismo , Humanos , Ratones , Ratones Transgénicos , Sistema Musculoesquelético/patología , Atención Posnatal , Manguito de los Rotadores/crecimiento & desarrollo , Manguito de los Rotadores/patología , Células Madre/metabolismo , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/patología , Tendones/crecimiento & desarrollo , Tendones/metabolismo , Tendones/patologíaRESUMEN
Although inflammation is necessary during the early phases of tissue repair, persistent inflammation contributes to fibrosis. Acute tendon injuries often heal through a fibrotic mechanism, which impedes regeneration and functional recovery. Because inflammation mediated by nuclear factor κB (NF-κB) signaling is implicated in this process, we examined the spatial, temporal, and cell type-specific activation profile of canonical NF-κB signaling during tendon healing. NF-κB signaling was maintained through all phases of tendon healing in mice, including the remodeling phase, and tenocytes and myofibroblasts from the Scleraxis (Scx) lineage were the predominant populations that retained NF-κB activation into the late stages of repair. We confirmed persistent NF-κB activation in myofibroblasts in human tendon scar tissue. Deleting the canonical NF-κB kinase, IKKß, in Scx-lineage cells in mice increased apoptosis and the deposition of the matrix protein periostin during the late stages of tendon repair, suggesting that persistent NF-κB signaling may facilitate myofibroblast survival and fibrotic progression. Consistent with this, myofibroblasts in human tendon scar samples displayed enhanced prosurvival signaling compared to control tissue. Together, these data suggest that NF-κB may contribute to fibrotic tendon healing through both inflammation-dependent and inflammation-independent functions, such as NF-κB-mediated cell survival.
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Miofibroblastos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Traumatismos de los Tendones/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ratones , Ratones Noqueados , Miofibroblastos/patología , FN-kappa B/genética , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/patologíaRESUMEN
BACKGROUND: Surgical repair of tendons is common, but function is often limited due to the formation of flexor tendon adhesions which reduce the mobility and use of the affected digit and hand. The severity of adhesion formation is dependent on numerous cellular processes many of which involve the actin cytoskeleton. Flightless I (Flii) is a highly conserved cytoskeletal protein, which has previously been identified as a potential target for improved healing of tendon injuries. Using human in vitro cell studies in conjunction with a murine model of partial laceration of the digital flexor tendon, we investigated the effect of modulating Flii levels on tenocyte function and formation of adhesions. METHODS: Human tenocyte proliferation and migration was determined using WST-1 and scratch wound assays following Flii knockdown by siRNA in vitro. Additionally, mice with normal and increased levels of Flii were subjected to a partial laceration of the digital flexor tendon in conjunction with a full tenotomy to immobilise the paw. Resulting adhesions were assessed using histology and immunohistochemistry for collagen I, III, TGF-ß1and -ß3 RESULTS: Flii knockdown significantly reduced human tenocyte proliferation and migration in vitro. Increasing the expression of Flii significantly reduced digital tendon adhesion formation in vivo which was confirmed through significantly smaller adhesion scores based on collagen fibre orientation, thickness, proximity to other fibres and crimping. Reduced adhesion formation was accompanied with significantly decreased deposition of type I collagen and increased expression of TGF-ß1 in vivo. CONCLUSIONS: These findings suggest that increasing the level of Flii in an injured tendon may be beneficial for decreasing tendon adhesion formation.
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Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/patología , Tenocitos/fisiología , Adherencias Tisulares/genética , Adherencias Tisulares/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Colágenos Asociados a Fibrillas/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica , Ratones Endogámicos BALB C , Ratones Transgénicos , Traumatismos de los Tendones/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Matriz Extracelular/genética , Traumatismos de los Tendones/genética , Factores de Transcripción/genética , Transcriptoma/genética , Animales , Colágeno/genética , Regulación de la Expresión Génica/genética , Caballos/genética , Caballos/crecimiento & desarrollo , ARN Mensajero/genética , RNA-Seq , Traumatismos de los Tendones/patología , Tendones/crecimiento & desarrollo , Tendones/patología , Tenocitos/metabolismo , Tenocitos/patología , Cicatrización de Heridas/genéticaRESUMEN
Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1ß. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1ß-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.
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Comunicación Celular/genética , Inflamación/genética , Interleucina-1beta/genética , Activación de Macrófagos/genética , Comunicación Celular/efectos de la radiación , Polaridad Celular/genética , Polaridad Celular/efectos de la radiación , Técnicas de Cocultivo , Campos Electromagnéticos , Humanos , Inflamación/inmunología , Inflamación/terapia , Macrófagos/inmunología , Macrófagos/metabolismo , Magnetoterapia , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/efectos de la radiación , Transducción de Señal , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/patología , Traumatismos de los Tendones/terapia , Tendones/metabolismo , Tendones/patología , Tendones/efectos de la radiación , Factor de Necrosis Tumoral alfa/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/efectos de la radiaciónRESUMEN
Achilles tendon healing (ATH) remains an unanswered question in the field of sports medicine because it does not produce tissue with homology to the previously uninjured tissue. Oestrogen receptor ß (ERß) is involved in the injury and repair processes of tendons. Our previous study confirmed that ERß plays a role in the early stage of ATH by affecting adipogenesis, but its role in extracellular matrix (ECM) remodelling is unknown. We established a 4-week Achilles tendon repair model to investigate the mechanism through which ERß affects ATH at the very beginning of ECM remodelling phase. In vitro studies were performed using tendon-derived stem cells (TDSCs) due to their promising role in tendon healing. Behavioural and biomechanical tests revealed that ERß-deficient mice exhibit weaker mobility and inferior biomechanical properties, and immunofluorescence staining and qRT-PCR showed that these mice exhibited an erroneous ECM composition, as mainly characterized by decreased collagen type I (Col I) deposition. The changes in gene expression profiles between ERß-knockout and WT mice at 1 week were analysed by RNA sequencing to identify factors affecting Col I deposition. The results highlighted the IRF5-CCL3 axis, and this finding was verified with CCL3-treated TDSCs. These findings revealed that ERß regulates Col I deposition during ATH via the IRF5-CCL3 axis.
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Quimiocina CCL3/genética , Receptor beta de Estrógeno/genética , Factores Reguladores del Interferón/genética , Traumatismos de los Tendones/terapia , Tendón Calcáneo/lesiones , Tendón Calcáneo/metabolismo , Tendón Calcáneo/patología , Adipogénesis/genética , Animales , Diferenciación Celular/genética , Colágeno Tipo I/genética , Modelos Animales de Enfermedad , Matriz Extracelular , Humanos , Masculino , Ratones , Ratones Noqueados , Medicina Deportiva , Trasplante de Células Madre , Traumatismos de los Tendones/genética , Traumatismos de los Tendones/patología , Cicatrización de Heridas/genéticaRESUMEN
Tendon injuries are common and have a high incidence of re-rupture that can cause loss of functionality. Therapies with adipose-derived stem cells (ASC) and the microcurrent (low-intensity electrical stimulation) application present promising effects on the tissue repair. We analyzed the expression of genes and the participation of some molecules potentially involved in the structural recovery of the Achilles tendon of rats, in response to the application of both therapies, isolated and combined. The tendons were distributed in five groups: normal (N), transected (T), transected and ASC (C) or microcurrent (M) or with ASC, and microcurrent (MC). Microcurrent therapy was beneficial for tendon repair, as it was observed a statistically significant increase in the organization of the collagen fibers, with involvement of the TNC, CTGF, FN, FMDO, and COL3A1 genes as well as PCNA, IL-10, and TNF-α. ASC therapy significantly increased the TNC and FMDO genes expression with no changes in the molecular organization of collagen. With the association of therapies, a significant greater collagen fibers organization was observed with involvement of the FMOD gene. The therapies did not affect the expression of COL1A1, SMAD2, SMAD3, MKX, and EGR1 genes, nor did they influence the amount of collagen I and III, caspase-3, tenomodulin (Tnmd), and hydroxyproline. In conclusion, the application of the microcurrent isolated or associated with ASC increased the organization of the collagen fibers, which can result in a greater biomechanical resistance in relation to the tendons treated only with ASC. Future studies will be needed to demonstrate the biological effects of these therapies on the functional recovery of injured tendons.