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Tendons transmit the muscle contraction forces to bones and drive joint movement throughout life. While extensive research have indicated the essentiality of mechanical forces on tendon development, a comprehensive understanding of the fundamental role of mechanical forces still needs to be impaerted. This scoping review aimed to summarize the current knowledge about the role of mechanical forces during the tendon developmental phase. The electronic database search using PubMed, performed in May 2023, yielded 651 articles, of which 16 met the prespecified inclusion criteria. We summarized and divided the methods to reduce the mechanical force into three groups: loss of muscle, muscle dysfunction, and weight‐bearing regulation. In contrast, there were few studies to analyze the increased mechanical force model. Most studies suggested that mechanical force has some roles in tendon development in the embryo to postnatal phase. However, we identified species variability and methodological heterogeneity to modulate mechanical force. To establish a comprehensive understanding, methodological commonality to modulate the mechanical force is needed in this field. Additionally, summarizing chronological changes in developmental processes across animal species helps to understand the essence of developmental tendon mechanobiology. We expect that the findings summarized in the current review serve as a groundwork for future study in the fields of tendon developmantal biology and mechanobiology. Key Findings: Recent mechano‐biological studies about tendon development have no consensus on the definition of mechanical force on the tendon. A clear definition of mechanical force based on species is needed to establish a comprehensive understanding of the role of mechanical force on tendon development. [ABSTRACT FROM AUTHOR]

Tendons and their attachment sites to bone, fibrocartilaginous tissues, have poor self-repair capacity when they rupture, and have risks of retear even after surgical repair. Thus, defining mechanisms underlying their repair is required in order to stimulate tendon repairing capacity. Here we used a rat surgical rotator cuff tear repair model and identified cells expressing the transcription factors Scleraxis (Scx) and SRY-box 9 (Sox9) as playing a crucial role in rotator cuff tendon-to-bone repair. Given the challenges of establishing stably reproducible models of surgical rotator cuff tear repair in mice, we newly established Scx-GFP transgenic rats in which Scx expression can be monitored by GFP. We observed tissue-specific GFP expression along tendons in developing ScxGFP transgenic rats and were able to successfully monitor tissue-specific Scx expression based on GFP signals. Among 3-, 6-, and 12-week-old ScxGFP rats, Scx+/Sox9+ cells were most abundant in 3-week-old rats near the site of humerus bone attachment to the rotator cuff tendon, while we observed significantly fewer cells in the same area in 6- or 12-week-old rats. We then applied a rotator cuff repair model using ScxGFP rats and observed the largest number of Scx+/Sox9+ cells at postoperative repair sites of 3-week-old relative to 6- or 12-week-old rats. Tendons attach to bone via fibrocartilaginous tissue, and cartilage-like tissue was seen at repair sites of 3-week-old but not 6- or 12-week-old rats during postoperative evaluation. Our findings suggest that Scx+/Sox9+ cells may function in rotator cuff repair, and that ScxGFP rats could serve as useful tools to develop therapies to promote rotator cuff repair by enabling analysis of these activities. • We newly established ScxGFP transgenic rats to detect Scx expression by GFP signals. • A surgical rotator cuff tear repair model was created in ScxGFP transgenic rats. • Scx+/Sox9+ cells were most abundant at repair site in 3- than 6- or 12-week-old rats. • Fibrocartilaginous tissue was induced at repair site in only 3-week-old rats. [ABSTRACT FROM AUTHOR]

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

Movement of the vertebrate body is supported by the connection of muscle, tendon and bone. Each skeletal muscle in the vertebrate body has a unique shape and attachment site; however, the mechanism that ensures reproducible muscle patterning is incompletely understood. In this study, we conducted targeted cell ablation using scleraxis (Scx)-Cre to examine the role of Scx-lineage cells in muscle morphogenesis and attachment in mouse embryos. We found that muscle bundle shapes and attachment sites were significantly altered in embryos with Scx-lineage cell ablation. Muscles in the forelimb showed impaired bundle separation and limb girdle muscles distally dislocated from their insertion sites. Scxlineage cells were required for post-fusion myofiber morphology, but not for the initial segregation of myoblasts in the limb bud. Furthermore, muscles could change their attachment site, even after formation of the insertion. Lineage tracing suggested that the muscle patterning defect was primarily attributed to the reduction of tendon/ligament cells. Our study demonstrates an essential role of Scx-lineage cells in the reproducibility of skeletal muscle attachment, in turn revealing a previously unappreciated tissue-tissue interaction in musculoskeletal morphogenesis. [ABSTRACT FROM AUTHOR]

The molecular cascade underlying tendon formation starts when progenitor cells begin to express the Scleraxis (Scx) gene. Scx knockout mice develop some but not all tendons, suggesting that additional factors are necessary for tendon commitment, maintenance, and differentiation. Other transcription factors, such as Mohawk (Mkx) or early growth response (Egr), maintain Scx expression and extracellular matrix formation during fibrillogenesis. The inhibition of wingless and int-related protein signaling is necessary and sufficient to induce the expression of Scx. Once the commitment of tenogenic lineage occurs, transforming growth factor-beta (TGFβ) induces the Scx gene expression, becoming involved in the maintenance of tendon cell fate. From this point of view, we discussed two phases of the tenogenic process during limb development: dependent and independent of mechanical forces. Finally, we highlight the importance of understanding embryonic tendon development to improve therapeutic strategies in regenerative medicines for tendons. [ABSTRACT FROM AUTHOR]

RAB23 is a small GTPase which functions at the plasma membrane to regulate growth factor signaling. Mutations in RAB23 cause Carpenter syndrome, a condition that affects normal organogenesis and patterning. In this study, we investigate the role of RAB23 in musculoskeletal development and show that it is required for patella bone formation and for the maintenance of tendon progenitors. The patella is the largest sesamoid bone in mammals and plays a critical role during movement by providing structural and mechanical support to the knee. Rab23−/− mice fail to form a patella and normal knee joint. The patella is formed from Sox9 and scleraxis (Scx) double-positive chondroprogenitor cells. We show that RAB23 is required for the specification of SOX9 and scleraxis double-positive patella chondroprogenitors during the formation of patella anlagen and the subsequent establishment of patellofemoral joint. We find that scleraxis and SOX9 expression are disrupted in Rab23−/− mice, and as a result, development of the quadriceps tendons, cruciate ligaments, patella tendons, and entheses is either abnormal or lost. TGFβ-BMP signaling is known to regulate patella initiation and patella progenitor differentiation and growth. We find that the expression of TGFβR2, BMPR1, BMP4, and pSmad are barely detectable in the future patella site and in the rudimentary tendons and ligaments around the patellofemoral joint in Rab23−/− mice. Also, we show that GLI1, SOX9, and scleraxis, which regulate entheses establishment and maturation, are weakly expressed in Rab23−/− mice. Further analysis of the skeletal phenotype of Rab23−/− mice showed a close resemblance to that of Tgfβ2−/− mice, highlighting a possible role for RAB23 in regulating TGFβ superfamily signaling.

Aged tendons have disrupted homeostasis, increased injury risk, and impaired healing capacity. Understanding mechanisms of homeostatic disruption is crucial for developing therapeutics to retain tendon health through the lifespan. Here, we developed a novel model of accelerated tendon extracellular matrix (ECM) aging via depletion of Scleraxis-lineage cells in young mice (Scx-DTR). Scx-DTR recapitulates many aspects of tendon aging including comparable declines in cellularity, alterations in ECM structure, organization, and composition. Single-cell RNA sequencing demonstrated a conserved decline in tenocytes associated with ECM biosynthesis in aged and Scx-DTR tendons, identifying the requirement for Scleraxis-lineage cells during homeostasis. However, the remaining cells in aged and Scx-DTR tendons demonstrate functional divergence. Aged tenocytes become pro-inflammatory and lose proteostasis. In contrast, tenocytes from Scx-DTR tendons demonstrate enhanced remodeling capacity. Collectively, this study defines Scx-DTR as a novel model of accelerated tendon ECM aging and identifies novel biological intervention points to maintain tendon function through the lifespan.

Intermuscular bones (IBs) are ossified from tendons and only occur in lower teleosts. Positive association between the regulation of scleraxis gene (scx) and tendon development gave us reasons to speculate that the scx gene may play a potential role in regulating the development of IBs. A phylogenetic analysis conducted for this study revealed potential functional differentiation between two scx orthologues, scxa and scxb. The scxa−/− and scxb−/− zebrafish were generated through CRISPR-Cas9 technology to study the role of scx in the IB and rib development. The results showed a significant reduction of the number of IBs in adult scxa-1-/- zebrafish, with almost 70% reduction (15–25 IBs) compared to the wild type scxa-1+/+ zebrafish (76–80 IBs). In the scxa-1+/+ adults, IBs were observed in both dorsal and tail segments; however, in scxa-1-/- fish IBs were observed only in the tail segment (none in the dorsal segment). Although scxa-1−/− zebrafish had rib defects, the mutants were viable and fertile as adult fish. The scxb−/− zebrafish had the same number of IBs and same skeletal phenotype as the wild-type fish. This suggests that only scxa has a crucial role in the IB development, and confirms functional differentiation of scx orthologues. To further clarify the molecular mechanism by which scxa affects the IB development, we conducted comparative transcriptome analysis of dorsal tissue samples of scxa-1+/+ (with IBs) and scxa-1−/− (without IBs), and further verified the expression of key genes via qPCR. This is the first study to identify a gene that controls the amount of IBs in fish, and it provides a new sight into the effects of scxa on the molecular mechanism of IB development in fish.

Herein, we investigate the high incidence of umbilical hernia and tippy-toe standing and their underlying changes in gene expression and proliferation in myostatin knockout (MSTN−/−) pigs. Thirty-six male MSTN−/− pigs were generated by somatic cell nuclear transfer (SCNT). These pigs presented a considerably high incidence of tippy-toe standing and umbilical hernia (69.4% and 61.1%, respectively). The tendon to body weight ratio was significantly lower than wild-type pigs (0.202 ± 0.017 vs 0.250 ± 0.004, respectively). The crimp length of the MSTN−/− tendon was significantly longer than that of wild-type pigs. The expression of MSTN and the activin type IIB (ACVR2B) was detected in the tendon and linea alba of MSTN−/− pigs. MSTN treatment significantly increased the phosphorylation of Smad2/3 in both tendon and linea alba fibroblasts. Type I collagen (Col1A) and Scleraxis (Scx) expression levels in the tendon and linea alba of MSTN−/− pigs were significantly lower than those in wild-type in vivo, whereas and cyclin-dependent kinase inhibitor 1 (p21) expression levels were higher. Treatment of tendon and linea alba fibroblasts with recombinant MSTN increased Col1A and Scx and decreased p21 expression in vivo. Moreover, there was a significant increase in fibroblast proliferation after treatment. The results indicated that MSTN regulates collagen expression and proliferation in tendon and linea alba fibroblasts; thus, MSTN deficiency causes collagen-related pathological features in MSTN−/− pigs. Hence, MSTN could be used as a therapeutic target for treating UH and tendon abnormalities. [ABSTRACT FROM AUTHOR]

The sclera is an ocular tissue rich of collagenous extracellular matrix, which is built up and maintained by relatively few, still poorly characterized fibroblast-like cells. The aims of this study are to add to the characterization of scleral fibroblasts and to examine the reaction of these fibroblasts to inflammatory stimulation in an ex vivo organotypic model. Scleras of scleraxis-GFP (SCX-GFP) mice were analyzed using immunohistochemistry and qRT-PCR for the expression of the tendon cell associated marker genes scleraxis (SCX), mohawk and tenomodulin. In organotypic tissue culture, explanted scleras of adult scleraxis GFP reporter mice were exposed to 10 ng/ml recombinant interleukin 1-ß (IL1-ß) and IL1-ß in combination with dexamethasone. The tissue was then analyzed by immunofluorescence staining of the inflammation- and fibrosis-associated proteins IL6, COX-2, iNOS, connective tissue growth factor, MMP2, MMP3, and MMP13 as well as for collagen fibre degradation using a Collagen Hybridizing Peptide (CHP) binding assay. The mouse sclera displayed a strong expression of scleraxis promoter-driven GFP, indicating a tendon cell-like phenotype, as well as expression of scleraxis, tenomodulin and mohawk mRNA. Upon IL1-ß stimulation, SCX-GFP+ cells significantly upregulated the expression of all proteins analysed. Moreover, IL1-ß stimulation resulted in significant collagen degradation. Adding the corticosteroid dexamethasone significantly reduced the response to IL1-ß stimulation. Collagen degradation was significantly enhanced in the IL1-ß group. Dexamethasone demonstrated a significant rescue effect. This work provides insights into the characteristics of scleral cells and establishes an ex vivo model of scleral inflammation. [ABSTRACT FROM AUTHOR]

The cell differentiation of the musculoskeletal system is highly coordinated during limb development. In the distal-most region of the limb, WNT and FGF released from the apical ectodermal ridge maintain mesenchymal cells in the undifferentiated stage. Once the cells stop receiving WNT and FGF, they respond to differentiation signals. Particularly during tendon development, mesenchymal cells enter the cell differentiation program once Scleraxis (Scx) gene expression occurs. Among the signals that trigger the cell differentiation programs, TGFb signaling has been found to be closely involved in tendon differentiation. However, whether Scx gene expression depends merely on TGFb signaling or other signals is still not fully understood. In the present study, considering that WNT/b-catenin is an inhibitory signal of cell differentiation, we speculated possible antagonistic or additive effects between canonical Wnt/b-catenin and TGFb/SMAD signaling pathways to control Scx gene expression. We found that the blockade of WNT/b-catenin promoted Scx gene expression. In contrast, the inhibition of TGFb/SMAD signaling did not maintain Scx gene expression. Interestingly, the blockade of both WNT/b-catenin and TGFb/SMAD signaling at the same time promoted Scx gene expression. Thus the inhibition of WNT/b-catenin signaling appears to be necessary and sufficient to induce Scx gene expression. [ABSTRACT FROM AUTHOR]

Despite the requirement for Scleraxis-lineage (ScxLin) cells during tendon development, the function of ScxLin cells during adult tendon repair, post-natal growth, and adult homeostasis have not been defined. Therefore, we inducibly depleted ScxLin cells (ScxLinDTR) prior to tendon injury and repair surgery and hypothesized that ScxLinDTR mice would exhibit functionally deficient healing compared to wild-type littermates. Surprisingly, depletion of ScxLin cells resulted in increased biomechanical properties without impairments in gliding function at 28 days post-repair, indicative of regeneration. RNA sequencing of day 28 post-repair tendons highlighted differences in matrix-related genes, cell motility, cytoskeletal organization, and metabolism. We also utilized ScxLinDTR mice to define the effects on post-natal tendon growth and adult tendon homeostasis and discovered that adult ScxLin cell depletion resulted in altered tendon collagen fibril diameter, density, and dispersion. Collectively, these findings enhance our fundamental understanding of tendon cell localization, function, and fate during healing, growth, and homeostasis.

TGFβ plays a critical role in tendon formation and healing. While its downstream effector Smad3 has been implicated in the healing process, little is known about the role of Smad3 in normal tendon development or tenocyte gene expression. Using mice deficient in Smad3 (Smad3(-/-) ), we show that Smad3 ablation disrupts tendon architecture and has a dramatic impact on normal gene and protein expression during development as well as in mature tendon. In developing and adult tendon, loss of Smad3 results in reduced protein expression of the matrix components Collagen 1 and Tenascin-C. Additionally, when compared to wild type, tendon from adult Smad3(-/-) mice shows a down regulation of key tendon marker genes. Finally, we have established that Smad3 has the ability to physically interact with the critical transcriptional regulators Scleraxis and Mohawk. Together these results indicate a central role for Smad3 in normal tendon formation and in the maintenance of mature tendon.

One of the main challenges relating to tendons is to understand the regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation have not been identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation, assessed with the expression of the tendon markers Scx, Col1a1 and Tnmd as compared to cells cultured on plastic substrate. The 3D-fibrin environment was more favorable for Scx and Col1a1 expression compared to 2D cultures. We also identified TGFβ2 as a negative regulator of Tnmd expression in C3H10T1/2 cells in 2D and 3D cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters upon which we could act to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells.

Studies of cell fate focus on specification, but little is known about maintenance of the differentiated state. In this study, we find that the mouse tendon cell fate requires continuous maintenance in vivo and identify an essential role for TGFβ signaling in maintenance of the tendon cell fate. To examine the role of TGFβ signaling in tenocyte function the TGFβ type II receptor (Tgfbr2) was targeted in the Scleraxis-expressing cell lineage using the ScxCre deletor. Tendon development was not disrupted in mutant embryos, but shortly after birth tenocytes lost differentiation markers and reverted to a more stem/progenitor state. Viral reintroduction of Tgfbr2 to mutants prevented and even rescued tenocyte dedifferentiation suggesting a continuous and cell autonomous role for TGFβ signaling in cell fate maintenance. These results uncover the critical importance of molecular pathways that maintain the differentiated cell fate and a key role for TGFβ signaling in these processes.

Abstract Background Tendinopathies are common and difficult to resolve due to the formation of scar tissue that reduces the mechanical integrity of the tissue, leading to frequent reinjury. Tenocytes respond to both excessive loading and unloading by producing pro-inflammatory mediators, suggesting that these cells are actively involved in the development of tendon degeneration. The transcription factor scleraxis (Scx) is required for the development of force-transmitting tendon during development and for mechanically stimulated tenogenesis of stem cells, but its function in adult tenocytes is less well-defined. The aim of this study was to further define the role of Scx in mediating the adult tenocyte mechanoresponse. Results Equine tenocytes exposed to siRNA targeting Scx or a control siRNA were maintained under cyclic mechanical strain before being submitted for RNA-seq analysis. Focal adhesions and extracellular matrix-receptor interaction were among the top gene networks downregulated in Scx knockdown tenocytes. Correspondingly, tenocytes exposed to Scx siRNA were significantly softer, with longer vinculin-containing focal adhesions, and an impaired ability to migrate on soft surfaces. Other pathways affected by Scx knockdown included increased oxidative phosphorylation and diseases caused by endoplasmic reticular stress, pointing to a larger role for Scx in maintaining tenocyte homeostasis. Conclusions Our study identifies several novel roles for Scx in adult tenocytes, which suggest that Scx facilitates mechanosensing by regulating the expression of several mechanosensitive focal adhesion proteins. Furthermore, we identified a number of other pathways and targets affected by Scx knockdown that have the potential to elucidate the role that tenocytes may play in the development of degenerative tendinopathy.

Tendons and ligaments are crucial components of the musculoskeletal system, yet the pathways specifying these fates remain poorly defined. Through a screen of known bioactive chemicals in zebrafish, we identified a new pathway regulating tendon cell induction. We established that statin, through inhibition of the mevalonate pathway, causes an expansion of the tendon progenitor population. Co-expression and live imaging studies indicate that the expansion does not involve an increase in cell proliferation, but rather results from re-specification of cells from the neural crest-derived sox9a+/sox10+ skeletal lineage. The effect on tendon cell expansion is specific to the geranylgeranylation branch of the mevalonate pathway and is mediated by inhibition of Rac activity. This work establishes a novel role for the mevalonate pathway and Rac activity in regulating specification of the tendon lineage. [ABSTRACT FROM AUTHOR]

Tendon and ligament injuries are not uncommon in clinics and have poor self-healing capacity due to their bloodless and slow-proliferative nature. Promoting the repair or reconstruction of an injured structure is an urgent problem. While Scleraxis (Scx) is a highly specific tendon cell marker, its function has not been explored to a large extent. Hence, Recombinant adenovirus was used to study the influence of Scx overexpression on directional differentiation of human amniotic mesenchymal stem cells (hMSCAs). hAMSCs modified with Scx could dramatically enhance the gene expression of tendon-related molecules, containing Scx, collagens I and III, Tenascin-C, fibronectin, matrix metalloproteinase-2 (MMP-2), lysyl oxidase-1 (LOX-1) and Tenomodulin at all-time points (P < 0.05), and the secretion of collagen I and III, fibronectin and Tenascin-C over time (P < 0.05) but did not impact the cell proliferation capacity (P > 0.05). Immunofluorescence staining showed the cobweb-like fusion of collagen I and fibronectin in the AdScx group on day 7, with higher average fluorescence intensity than the control (P < 0.05). After mixing with Matrigel, transplants were subcutaneously implanted in nude mice, obvious inflammation and rejection of immune response were not observed and HE staining showed a histological feature of swirl of fibers is closely linked in parallel in hAMSCs modified with Scx. On the contrary, in the control group, an unorganized connective structure with cell distributed randomly was spotted. The results of promoted directional differentiation of stem cells and the spatial structure of the normal tendon tissue in three-dimensional space manifested that Scx can be used as a specific marker for tendon cells, and as a positive regulator for directional differentiation of hAMSCs, which is possible to be applied to novel therapeutics for clinical tendon and ligament injury by hAMSCs modified with Scx. [ABSTRACT FROM AUTHOR]

BACKGROUND: Human amniotic mesenchymal stem cells have a wide variety of sources, low immunogenicity, and multilineage differentiation potential. Studies have confirmed that Scleraxis gene can induce human amniotic mesenchymal stem cells to differentiate into ligaments and accelerate tendon-bone healing. OBJECTIVE: To explore whether Scleraxis induces human amniotic mesenchymal stem cells to promote tendon-bone healing in vivo in rabbits, providing new options for clinical treatment of tendon-bone healing. METHODS: The study protocol was approved by the Ethic Committee of the Affiliated Hospital of Zunyi Medical University, and written informed consent was obtained from each puerpera. The healthy full-term maternal placenta was taken and cultured, and human amniotic mesenchymal stem cells were isolated and cultured by trypsin digestion twice. Then the morphology of the cells was observed under an inverted microscope, and the cells were further cultured until the third generation for subsequent experiments. The lentivirus carrying the Scleraxis gene was transfected into human amniotic mesenchymal stem cells in vitro. Expression levels of ligament-related genes were detected by real-time fluorescent quantitative PCR, and the expression levels of related proteins were detected by immunofluorescence. Human amniotic mesenchymal stem cells transfected with Scleraxis gene were injected into the extraarticular tendon-bone model of rats. After 3 months, specimens were taken to observe the tendon-bone healing. RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells from passage to third generation showed long fusiform and vortex-like adherent growth under the inverted phase contrast microscope. (2) The third-generation human amniotic mesenchymal stem cells expressed green fluorescence after 24 hours of infection with the Scleraxis gene lentivirus, and the fluorescence expression was strong and stable. (3) Cell counting kit-8 findings indicated that lentivirus transfection of Scleraxis gene showed no influence on the cell growth rate. (4) Real-time fluorescent quantitative PCR findings showed that the mRNA expression of Scleraxis and ligament-related genes type I collagen, type III collagen, Fibronectin and Tenascin-C was significantly increased after lentivirus transfection of Scleraxis gene. (5) The results of immunofluorescence showed that the expression levels of ligament-related proteins type I collagen, type III collagen, Fibronectin and Tenascin-C were increased after lentivirus transfection of Scleraxis gene. To conclude, in vivo animal experiments have confirmed that the lentivirus transfection of Scleraxis gene can accelerate the tendon-bone healing of the rabbit extraarticular tendon-bone model. [ABSTRACT FROM AUTHOR]

BACKGROUND: Acellular amniotic membrane scaffold is a natural scaffold with good biocompatibility, which has been widely used in tissue engineering. Scleraxis can promote the differentiation of human amniotic mesenchymal stem cells into human ligament cells and promote tendon-bone healing. OBJECTIVE: To explore whether acellular amniotic membrane scaffold combined with human amniotic mesenchymal stem cells transfected with Scleraxis can promote rabbit tendon-bone healing. METHODS: (1) Human amniotic mesenchymal stem cells were isolated and cultured in vitro. After passaged, the cell morphology was observed. (2) The Scleraxis lentivirus was constructed in vitro and then transfected into passage 3 human amniotic mesenchymal stem cells with optimal multiplicity of infection. The transfection efficiency was detected by q-PCR. (3) The acellular amniotic membrane scaffold was prepared by enzymatic digestion. Then the Scleraxis lentivirus-transfected cells were seeded on the acellular amniotic membrane scaffold in vitro. The cell growth on the scaffold was observed by phalloidin staining. (4) The New Zealand white rabbit tendon was covered with the acellular amniotic membrane scaffold combined with human amniotic mesenchymal stem cells transfected with Scleraxis lentivirus, followed by implanted into the bone tunnel. The tendon-bone healing was detected. RESULTS AND CONCLUSION: The passage 3 human amniotic mesenchymal stem cells adhered well. (2) After transfected with Scleraxis lentivirus for 96 hours, stable green fluorescence was observed. The mRNA expression level of Sclerxis was significantly increased, indicating a success transfection. The epithelial cells of the acellular amniotic membrane scaffold disappeared, indicating a relatively complete decellularization. The basal layer remained intact, and the extracellular matrix component still existed. Phalloidin staining results revealed that the cells on the acellular amniotic membrane scaffold were in good adhesion and growth, and the cell proliferation was not affected. Therefore, In vivo experimental results reveal that human acellular amniotic scaffold combined with human amniotic mesenchymal stem cells transfected with Scleraxis lentivirus can promote the tendon-bone healing. [ABSTRACT FROM AUTHOR]

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues. [ABSTRACT FROM AUTHOR]

The musculoskeletal system comprises muscles, tendons, ligaments, and bones. The connection site between muscle and tendon is termed "myotendinous junction," while the junction between tendon/ligament and bone is termed "enthesis." These two regions are the center of physical function, but how this functional complex is formed during development is unclear. In this review, we discussed recent findings about the development of tissues constituting the musculoskeletal system and the interactions among these tissues during development. The musculoskeletal system of the head develops in the mid-embryonic stage. In addition, head mesoderm-derived cells (muscle anlagen) and cranial neural crest cells (tendon and bone anlagen) interact with each other. Myogenesis initiates in the head without difficulty, even in the absence of cranial neural crest cells; however, muscle tissue does not grow under these conditions and remains small. Tendons, which differentiate from cranial neural crest cells, form myotendinous junctions at the stage at which desmin accumulates in the tendon-side muscle stump, leading to morphological maturation. Therefore, individual tissues (i.e., muscles, tendons, ligaments, and bones) constituting the musculoskeletal system form a functionally important complex, while mutually influencing one another. [ABSTRACT FROM AUTHOR]

Abstract Background A body of evidence demonstrating changes to the glutaminergic system in tendinopathy has recently emerged. This hypothesis was further tested by studying the effects of glutamate on the tenocyte phenotype, and the impact of loading and exposure to glucocorticoids on the glutamate signaling machinery. Methods Plantaris tendon tissue and cultured plantaris tendon derived cells were immunohisto-/cytochemically stained for glutamate, N-Methyl-D-Aspartate receptor 1 (NMDAR1) and vesicular glutamate transporter 2 (VGluT2). Primary cells were exposed to glutamate or receptor agonist NMDA. Cell death/viability was measured via LDH/MTS assays, and Western blot for cleaved caspase 3 (c-caspase 3) and cleaved poly (ADP-ribose) polymerase (c-PARP). Scleraxis mRNA (Scx)/protein(SCX) were analyzed by qPCR and Western blot, respectively. A FlexCell system was used to apply cyclic strain. The effect of glucocorticoids was studies by adding dexamethasone (Dex). The mRNA of the glutamate synthesizing enzymes Got1 and Gls, and NMDAR1 protein were measured. Levels of free glutamate were determined by a colorimetric assay. Results Immunoreactions for glutamate, VGluT2, and NMDAR1 were found in tenocytes and peritendinous cells in tissue sections and in cultured cells. Cell death was induced by high concentrations of glutamate but not by NMDA. Scleraxis mRNA/protein was down-regulated in response to NMDA/glutamate stimulation. Cyclic strain increased, and Dex decreased, Gls and Got1 mRNA expression. Free glutamate levels were lower after Dex exposure. Conclusions In conclusion, NMDA receptor stimulation leads to a reduction of scleraxis expression that may be involved in a change of phenotype in tendon cells. Glutamate synthesis is increased in tendon cells in response to strain and decreased by glucocorticoid stimulation. This implies that locally produced glutamate could be involved in the tissue changes observed in tendinopathy.

Klotho is an anti-aging gene that shortens the life span when disrupted and extends the lifespan when overexpressed. This study investigated whether autophagy plays a role in Klotho gene deficiency-induced arterial stiffening and hypertension. Klotho mutant heterozygous (KL+/−) mice and age- and sex-matched wild-type (WT) mice were used. Arteries were examined for autophagy using Western blot assays. Pulse wave velocity (PWV), a direct measure of arterial stiffness, and blood pressure (BP) increased significantly in KL (+/−) mice. The autophagy level, as measured by LC3-II expression and autophagy flux, increased in aortas of KL (+/−) mice, indicating that Klotho gene deficiency upregulated autophagy. Chloroquine diminished Klotho gene deficiency-induced increases in PWV and BP and eliminated the upregulation of autophagic flux in KL (+/−) mice. Klotho gene deficiency-induced arterial stiffness was accompanied by upregulation of MMP9, TGFβ-1, TGFβ-3, RUNX2, and ALP, but these changes were effectively mitigated by chloroquine. Chloroquine also halted an increase in scleraxis expression in aortas of Klotho (+/−) mice. In cultured mouse aortic smooth muscle cells, Klotho gene deficiency increased autophagy, leading to upregulation of scleraxis, a key transcription factor of collagen synthesis. Klotho gene deficiency failed to upregulate scleraxis expression when autophagy was inhibited, suggesting that autophagy is a critical mediator of Klotho gene deficiency-induced upregulation of scleraxis. Suppression of enhanced autophagy by chloroquine lessens Klotho gene deficiency-induced arterial stiffening and hypertension by stopping upregulation of MMP9 and scleraxis. The enhanced autophagic activity plays a crucial role in Klotho gene deficiency-induced arterial stiffening and hypertension. Key messages: Klotho gene deficiency upregulates autophagy. Upregulation of autophagy plays a role in the pathogenesis of arterial stiffening. Autophagy regulates MMP9 activity and scleraxis expression. [ABSTRACT FROM AUTHOR]

BACKGROUND: Human amniotic mesenchymal stem cells have multi-directional differentiation ability. Studies have shown that both Scleraxis and basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts. OBJECTIVE: To explore whether Scleraxis combined with basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts and to observe their differentiation effects. METHODS: The study protocol was approved by the ethic committee of the Affiliated Hospital of Zunyi Medical University, and written informed consent was obtained from each puerpera. The amniotic membrane from the full-term placenta was separated, and human amniotic mesenchymal stem cells were isolated by a two-step enzyme digestion. The morphology of the cells was observed by inverted phase contrast microscope. The structure of fresh human amniotic membrane was observed by hematoxylin-eosin staining. The third generation of human amniotic mesenchymal stem cells were cultured in four groups: (1) normally cultured human amnion mesenchymal stem cell culture (simple culture group); (2) human amniotic mesenchymal stem cells infected with Scleraxis gene lentivirus (Scleraxis group); (3) human amniotic mesenchymal stem cells induced by basic fibroblast growth factor (basic fibroblast growth factor group); (4) human amniotic mesenchymal stem cells induced by Scleraxis and basic fibroblast growth factor (combined induction group). The proliferation ability of the cells in each group was detected by cell counting kit-8 method at 3 days of cell culture. Real-time quantitative PCR was performed to evaluate the cell differentiation into human ligament fibroblasts in each group at 14 days of culture. RESULTS AND CONCLUSION: (1) Under the inverted phase contrast microscope, the third-generation human amniotic mesenchymal stem cells were long-fusiform and exhibited vortex-like adherent growth. (2) Under the inverted phase contrast microscope, the fresh human amniotic membrane had an obvious layered structure, which was divided into five layers: epithelial layer, basal layer, dense layer, fibroblast layer and sponge layer. (3) Human amniotic mesenchymal stem cells strongly and stably expressed green fluorescence at 24 hours after infection with Scleraxis gene lentivirus. The morphology of human amniotic mesenchymal stem cells induced by basic fibroblast growth factor was changed, and after 14 days of induction its morphology was similar to that of human ligament fibroblasts. (4) The results of cell counting kit-8 showed that the four groups of cells showed a S-type growth. The Scleraxis group, basic fibroblast growth factor group, combined induction group had stronger proliferation ability than the simple culture group, but there was no significant difference in the proliferative capacity between the three groups. (5) Real-time fluorescent quantitative PCR showed that the mRNA expression levels of ligament-related genes type I collagen, type III collagen, Fibronectin, Tenascin-C and TNMD in the Scleraxis group, basic fibroblast growth factor group, and combined induction group were significantly higher than those in the simple culture group (P < 0.05). The mRNA expression levels of ligament-related genes in the combined induction group were significantly higher than those in the Slckleris group and basic fibroblast growth factor group (P < 0.05). These results indicate that the combination of Scleraxis gene and basic fibroblast growth factor can promote the differentiation of human amniotic mesenchymal stem cells into human ligament fibroblasts, providing a new idea for the treatment of ligament injury. [ABSTRACT FROM AUTHOR]

The transcription factor scleraxis (Scx) is required for tendon development; however, the function of Scx is not fully understood. Although Scx is expressed by all tendon progenitors and cells, only long tendons are disrupted in the Scx-/- mutant; short tendons appear normal and the ability of muscle to attach to skeleton is not affected. We recently demonstrated that long tendons are formed in two stages: first, by muscle anchoring to skeleton via a short tendon anlage; and second, by rapid elongation of the tendon in parallel with skeletal growth. Through lineage tracing, we extend these observations to all long tendons and show that tendon elongation is fueled by recruitment of new mesenchymal progenitors. Conditional loss of Scx in mesenchymal progenitors did not affect the first stage of anchoring; however, new cells were not recruited during elongation and long tendon formation was impaired. Interestingly, for tenocyte recruitment, Scx expression was required only in the recruited cells and not in the recruiting tendon. The phenotype of Scx mutants can thus be understood as a failure of tendon cell recruitment during tendon elongation. [ABSTRACT FROM AUTHOR]

Background: The effects of fibroblast growth factor 2 (FGF-2) on healing after surgical repair of chronic rotator cuff (RC) tears remain unclear. Hypothesis: FGF-2 enhances tenogenic healing response, leading to biomechanical and histological improvement of repaired chronic RC tears in rats. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (n = 117) underwent unilateral surgery to refix the supraspinatus tendon to its insertion site 3 weeks after detachment. Animals were assigned to either the FGF-2 group or a control group. The effects of FGF-2 were assessed via biomechanical tests at 3 weeks after detachment and at 6 and 12 weeks postoperatively and were assessed histologically and immunohistochemically for proliferating cell nuclear antigen and mesenchymal stem cell (MSC)–related markers at 2, 6, and 12 weeks postoperatively. The expression of tendon/enthesis-related markers, including SRY-box 9 (Sox9), scleraxis (Scx), and tenomodulin (Tnmd), were assessed by real-time reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. The effect of FGF-2 on comprehensive gene expressions at the healing site was evaluated by microarray analysis. Results: The FGF-2 group showed a significant increase in mechanical strength at 6 and 12 weeks compared with control; the FGF-2 group also showed significantly higher histological scores at 12 weeks than control, indicating the presence of more mature tendon-like tissue. At 12 weeks, Scx and Tnmd expression increased significantly in the FGF-2 group, whereas no significant differences in Sox9 were found between groups over time. At 2 weeks, the percentage of positive cells expressing MSC-related markers increased in the FGF-2 group. Microarray analysis at 2 weeks after surgery showed that the expression of several growth factor genes and extracellular matrix–related genes was influenced by FGF-2 treatment. Conclusion: FGF-2 enhanced the formation of tough tendon-like tissues including an increase in Scx - or Tnmd -expressing cells at 12 weeks after surgical repair of chronic RC tears. The increase in mesenchymal progenitors and the changes in gene expression upon FGF-2 treatment in the early phase of healing appear to be related to a certain favorable microenvironment for tenogenic healing response of chronic RC tears. Clinical Relevance: These findings may provide advantages in therapeutic strategies for patients with RC tears. [ABSTRACT FROM AUTHOR]

Tendons and ligaments are dense fibrous bands of connective tissue that integrate musculoskeletal components in vertebrates. Tendons connect skeletal muscles to the bone and function as mechanical force transmitters, whereas ligaments bind adjacent bones together to stabilize joints and restrict unwanted joint movement. Fibroblasts residing in tendons and ligaments are called tenocytes and ligamentocytes, respectively. Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that is expressed at high levels in tenocytes and ligamentocytes, and is also present in periodontal ligament cells and tendon stem/progenitor cells. Tnmd is related to chondromodulin-1 (Chm1), a cartilage-derived angiogenesis inhibitor, and both Tnmd and Chm1 are expressed in the CD31− avascular mesenchyme. The conserved C-terminal hydrophobic domain of these proteins, which is characterized by the eight Cys residues to form four disulfide bonds, may have an anti-angiogenic function. This review highlights the molecular characterization and function of Tnmd, a specific marker of tendons and ligaments.

The equine periodontal ligament (PDL) is a fibrous connective tissue that covers the intra-alveolar parts of the tooth and anchors it to the alveolar bone—it, therefore, provides a similar function to a tendinous structure. While several studies have considered the formation and structure of tendons, there is insufficient information particularly on the molecular composition of the PDL. Especially for the equine PDL, there is limited knowledge concerning the expression of genes commonly regarded as typical for tendon tissue. In this study, the gene expression of, e.g., collagen type 1 alpha 1 (COL1), collagen type 3 alpha 1 (COL3), scleraxis (SCX), and fibrocartilage markers was examined in the functional mature equine PDL compared with immature and mature equine tendon tissue. PDL samples were obtained from incisor, premolar, and molar teeth from seven adult horses. Additionally, tendon samples were collected from four adult horses and five foals at different sampling locations. Analyses of gene expression were performed using real-time quantitative polymerase chain reaction (qRT-PCR). Significantly higher expression levels of COL1 and 3 were found in the mature equine PDL in comparison with mature tendon, indicating higher rates of collagen production and turnover in the mature equine PDL. The expression levels of SCX, a specific marker for tenogenic-differentiated cells, were on a similar level in functional mature PDL and in mature tendon tissue. Evidence of chondrogenic metaplasia, often found in tendon entheses or in pressurized regions of tendons, was not found in the mature equine PDL. The obtained results justify further experiments focused on the possible use of equine PDL cells for cell-based regenerative therapies.

Background: Tendinopathies are common and difficult to resolve due to the formation of scar tissue that reduces the mechanical integrity of the tissue, leading to frequent reinjury. Tenocytes respond to both excessive loading and unloading by producing pro-inflammatory mediators, suggesting that these cells are actively involved in the development of tendon degeneration. The transcription factor scleraxis (Scx) is required for the development of force-transmitting tendon during development and for mechanically stimulated tenogenesis of stem cells, but its function in adult tenocytes is less well-defined. The aim of this study was to further define the role of Scx in mediating the adult tenocyte mechanoresponse. Results: Equine tenocytes exposed to siRNA targeting Scx or a control siRNA were maintained under cyclic mechanical strain before being submitted for RNA-seq analysis. Focal adhesions and extracellular matrix-receptor interaction were among the top gene networks downregulated in Scx knockdown tenocytes. Correspondingly, tenocytes exposed to Scx siRNA were significantly softer, with longer vinculin-containing focal adhesions, and an impaired ability to migrate on soft surfaces. Other pathways affected by Scx knockdown included increased oxidative phosphorylation and diseases caused by endoplasmic reticular stress, pointing to a larger role for Scx in maintaining tenocyte homeostasis. Conclusions: Our study identifies several novel roles for Scx in adult tenocytes, which suggest that Scx facilitates mechanosensing by regulating the expression of several mechanosensitive focal adhesion proteins. Furthermore, we identified a number of other pathways and targets affected by Scx knockdown that have the potential to elucidate the role that tenocytes may play in the development of degenerative tendinopathy. [ABSTRACT FROM AUTHOR]

Tendon stem/progenitor cells (TSPCs) have been identified as a rare population in tendons. In vitro propagation is indispensable to obtain sufficient quantities of TSPCs for therapies. However, culture-expanded TSPCs are prone to lose their phenotype, resulting in an inferior repaired capability. And little is known about the underlying mechanism. Here, we found that altered gene expression was associated with increased histone deacetylase (HDAC) activity and expression of HDAC subtypes. Therefore, we exposed ScxGFP mice-derived TSPCs to HDAC inhibitor (HDACi) trichostatin A (TSA) or valproic acid (VPA), and observed significant expansion of ScxGFP + cells without altering phenotypic properties. TSA upregulated Scx expression by inhibiting HDAC1 and -3, and increasing the H3K27Ac level of Tgfb1 and - 2 genome region. Additionally, cell sheets formed from TSA-pretreated mTSPCs retained the ability to accelerate tendon repair in vivo . Thus, our results uncovered an unrecognized role of HDACi in phenotypic and functional mTSPCs expansion to enhance their therapeutic potential. [ABSTRACT FROM AUTHOR]

Put most simply, heterotopic ossification (HO) is the abnormal formation of bone at extraskeletal sites. HO can be classified into two main subtypes, genetic and acquired. Acquired HO is a common complication of major connective tissue injury, traumatic central nervous system injury, and surgical interventions, where it can cause significant pain and postoperative disability. A particularly devastating form of HO is manifested in the rare genetic disorder, fibrodysplasia ossificans progressiva (FOP), in which progressive heterotopic bone formation occurs throughout life, resulting in painful and disabling cumulative immobility. While the central role of stem/progenitor cell populations in HO is firmly established, the identity of the offending cell type(s) remains to be conclusively determined, and little is known of the mechanisms that direct these progenitor cells to initiate cartilage and bone formation. In this review, we summarize current knowledge of the cells responsible for acquired HO and FOP, highlighting the strengths and weaknesses of animal models used to interrogate the cellular origins of HO. [ABSTRACT FROM AUTHOR]

BACKGROUND: Tendon-to-bone healing is a complex and slow process, which often hinders the therapeutic outcomes. To enhance the tendon-to-bone healing, increasing cell bioactivity on the contact surface is a new strategy. Hypoxia-inducible factor 1α (HIF-1α) can induce the formation of blood vessel and bone tissue via many ways. However, it is unclear whether HIF-1α can strengthen differentiation of human amniotic mesenchymal stem cells (hAMSCs) induced by Scleraxis, a specific marker of tendon, and can be used in tendon and bone repair. OBJECTIVE: To investigate the ability of HIF-1α in enhancing Scleraxis to modify hAMSCs aiming to promote tendon-to-bone healing and its molecular mechanism. METHODS: Passage 3 hAMSCs were infected with AdHIF-1α, AdScx, AdGFP and AdHIF-1α+AdScx. The expressions of related genes of tendon, cartilage and bone tissue were detected at 3 and 7 days after infection. RESULTS AND CONCLUSION: Under the inverted phase contrast microscopy, the passage 3 hAMSCs were in spindle shape and presented with vortex-like adherent growth. Under the transmission electron microscopy, hAMSCs were in oval shape and had clear structure, with abundant endoplasmic reticulum and mitochondria. Fluorescence photographs were taken at 24 hours after adenovirus infection, showing about 50% red fluorescence expression in the AdHIF-1α group, while about 70% green fluorescence expression in the AdScx and AdGFP groups. Real time-PCR results showed that at 3 and 7 days after infection, the mRNA expressions of collagen type I, Fibronectin, RUNX2, VEGF and ALP in the AdHIF-1α+AdScx group, AdHIF-1α group and AdScx group were higher than those in the AdGFP group (P < 0.05); the mRNA expressions of collagen type I, Fibronectin, RUNX2, VEGF and ALP in the AdHIF-1α+AdScx group were significantly higher than those in the AdScx group (P < 0.05). Fluorescence immunohistochemistry results showed that the expression of collagen type I in the AdHIF-1α+AdScx group at 7 days after infection was higher than that at 3 days after infection. To conclude, HIF-1α can enhance the ability of Scleraxis to modify hAMSCs to promote tendon-to-bone healing by upregulating the expressions of molecular markers of tenocytes, chondrocytes and osteocytes. [ABSTRACT FROM AUTHOR]

Heart valves are complex structures composed of a heterogeneous population of valve interstitial cells (VICs), an overlying endothelium and highly organized layers of extracellular matrix. Alterations in valve homeostasis are characteristic of dysfunction and disease, however the mechanisms that initiate and promote valve pathology are poorly understood. Advancements have been largely hindered by the limited availability of tools for gene targeting in heart valve structures during embryogenesis and after birth. We have previously shown that the transcription factors Sox9 and Scleraxis (Scx) are required for heart valve formation and in this study we describe the recombination patterns of Sox9- and Scx-Cre lines at differential time points in aortic and mitral valve structures. In ScxCre; ROSA26GFP mice, recombination is undetected in valve endothelial cells (VECs) and low in VICs during embryogenesis. However, recombination increases in VICs from post natal stages and by 4 weeks side-specific patterns are observed. Using the inducible Sox9CreERT2 system, we observe recombination in VECs and VICs in the embryo, and high levels are maintained through post natal and juvenile stages. These Cre-drivers provide the field with new tools for gene targeting in valve cell lineages during differential stages of embryonic and post natal maturation and maintenance.