Exercise-induced Musclin determines the fate of fibro-adipogenic progenitors to control muscle homeostasis.

The effects of exercise on fibro-adipogenic progenitors (FAPs) are unclear, and the direct molecular link is still unknown. In this study, we reveal that exercise reduces the frequency of FAPs and attenuates collagen deposition and adipose formation in injured or disused muscles through Musclin. Mechanistically, Musclin inhibits FAP proliferation and promotes apoptosis in FAPs by upregulating FILIP1L. Chromatin immunoprecipitation (ChIP)-qPCR confirms that FoxO3a is the transcription factor of FILIP1L. In addition, the Musclin/FILIP1L pathway facilitates the phagocytosis of apoptotic FAPs by macrophages through downregulating the expression of CD47. Genetic ablation of FILIP1L in FAPs abolishes the effects of exercise or Musclin on FAPs and the benefits on the reduction of fibrosis and fatty infiltration. Overall, exercise forms a microenvironment of myokines in muscle and prevents the abnormal accumulation of FAPs in a Musclin/FILIP1L-dependent manner. The administration of exogenous Musclin exerts a therapeutic effect, demonstrating a potential therapeutic approach for muscle atrophy or acute muscle injury.

Integrative single-cell RNA and ATAC sequencing reveals that the FOXO1-PRDX2-TNF axis regulates tendinopathy.

Introduction: Tendinopathy, the most common form of chronic tendon disorder, leads to persistent tendon pain and loss of function. Profiling the heterogeneous cellular composition in the tendon microenvironment helps to elucidate rational molecular mechanisms of tendinopathy. Methods and results: In this study, through a multi-modal analysis, a single-cell RNA- and ATAC-seq integrated tendinopathy landscape was generated for the first time. We found that a specific cell subpopulation with low PRDX2 expression exhibited a higher level of inflammation, lower proliferation and migration ability, which not only promoted tendon injury but also led to microenvironment deterioration. Mechanistically, a motif enrichment analysis of chromatin accessibility showed that FOXO1 was an upstream regulator of PRDX2 transcription, and we confirmed that functional blockade of FOXO1 activity induced PRDX2 silencing. The TNF signaling pathway was significantly activated in the PRDX2-low group, and TNF inhibition effectively restored diseased cell degradation. Discussion: We revealed an essential role of diseased cells in tendinopathy and proposed the FOXO1-PRDX2-TNF axis is a potential regulatory mechanism for the treatment of tendinopathy.

Single-Cell Profiling of Tumor Microenvironment Heterogeneity in Osteosarcoma Identifies a Highly Invasive Subcluster for Predicting Prognosis.

Osteosarcoma is the most common malignant bone tumor in adolescents, and metastasis is the key reason for treatment failure and poor prognosis. Once metastasis occurs, the 5-year survival rate is only approximately 20%, and assessing and predicting the risk of osteosarcoma metastasis are still difficult tasks. In this study, cellular communication between tumor cells and nontumor cells was identified through comprehensive analysis of osteosarcoma single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data, illustrating the complex regulatory network in the osteosarcoma microenvironment. In line with the heterogeneity of osteosarcoma, we found subpopulations of osteosarcoma cells that highly expressed COL6A1, COL6A3 and MIF and were closely associated with lung metastasis. Then, BCDEG, a reliable risk regression model that could accurately assess the metastasis risk and prognosis of patients, was established, providing a new strategy for the diagnosis and treatment of osteosarcoma.

Histologic and biomechanical evaluation of the thoracolumbar fascia graft for massive rotator cuff tears in a rat model.

BACKGROUND: Fascial autografts, which are easily available grafts, have provided a promising option in patients with massive rotator cuff tears. However, no fascial autografts other than the fascia lata have been reported, and the exact healing process of the fascia-to-bone interface is not well understood. The objective of this study is to histologically and biomechanically evaluate the effect of the thoracolumbar fascia (TLF) on fascia-to-bone healing. METHODS: A total of 88 rats were used in this study. Eight rats were killed at the beginning to form an intact control group, and the other rats were divided randomly into 2 groups (40 rats per group): the TLF augmentation group (TLF group) and the repair group (R group). The right supraspinatus was detached, and a 3 × 5 mm defect of the supraspinatus was created. The TLF was used to augment the torn supraspinatus in the TLF group, whereas in the R group, the torn supraspinatus was repaired in only a transosseous manner. Histology and biomechanics were assessed at 1, 2, 4, 8, and 16 weeks postoperatively. RESULTS: The modified tendon maturation score of the TLF group was higher than that of the R group at 8 weeks (23.00 ± 0.71 vs. 24.40 ± 0.89, P = .025) and 16 weeks (24.60 ± 0.55 vs. 26.40 ± 0.55, P ≤ .001). The TLF group showed a rapid vascular reaction, and the peak value appeared at 1 week. Later, the capillary density decreased, and almost no angiogenesis was observed at 8 weeks postoperatively. Immunohistochemistry results demonstrated a significantly higher percentage of collagen I in the TLF group at 4, 8, and 16 weeks (24.78% ± 2.76% vs. 20.67% ± 2.11% at 4 weeks, P = .046; 25.46% ± 1.77% vs. 21.49% ± 2.33% at 8 weeks, P = .026; 34.77% ± 2.25% vs. 30.01% ± 3.17% at 16 weeks, P = .040) postoperatively. Biomechanical tests revealed that the ultimate failure force in the TLF group was significantly higher than that in the R group at the final evaluation (29.13 ± 2.49 N vs. 23.10 ± 3.47 N, P = .022). CONCLUSIONS: The TLF autograft can promote a faster biological healing process and a better fixation strength. It could be used as an alternative reinforcement or bridging patch when the fascia lata is not appropriate or available for superior capsule reconstruction (SCR).

Effects of aging on the histology and biochemistry of rat tendon healing.

INTRODUCTION: Tendon diseases and injuries are a serious problem for the aged population, often leading to pain, disability and a significant decline in quality of life. The purpose of this study was to determine the influence of aging on biochemistry and histology during tendon healing and to provide a new strategy for improving tendon healing. METHOD: A total of 24 Sprague-Dawley rats were equally divided into a young and an aged group. A rat patellar tendon defect model was used in this study. Tendon samples were collected at weeks 2 and 4, and hematoxylin-eosin, alcian blue and immunofluorescence staining were performed for histological analysis. Meanwhile, reverse transcription-polymerase chain reaction (RT-PCR) and western blot were performed to evaluate the biochemical changes. RESULTS: The histological scores in aged rats were significantly lower than those in young rats. At the protein level, collagen synthesis-related markers Col-3, Matrix metalloproteinase-1 and Metallopeptidase Inhibitor 1(TIMP-1) were decreased at week 4 in aged rats compared with those of young rats. Though there was a decrease in the expression of the chondrogenic marker aggrecan at the protein level in aged tendon, the Micro-CT results from weeks 4 samples showed no significant difference(p>0.05) on the ectopic ossification between groups. Moreover, we found more adipocytes accumulated in the aged tendon defect with the Oil Red O staining and at the gene and protein levels the markers related to adipogenic differentiation. CONCLUSIONS: Our findings indicate that tendon healing is impaired in aged rats and is characterized by a significantly lower histological score, decreased collagen synthesis and more adipocyte accumulation in patellar tendon after repair.

Exosomes Derived from Bone Marrow Stromal Cells (BMSCs) Enhance Tendon-Bone Healing by Regulating Macrophage Polarization.

BACKGROUND Inflammation after tendon-bone junction injury results in the formation of excessive scar tissue and poor biomechanical properties. Recent research has shown that exosomes derived from bone marrow stromal cells (BMSCs) can modulate inflammation during tissue healing. Thus, our study aimed to enhance tendon-bone healing by use of BMSC-derived exosomes (BMSC-Exos). MATERIAL AND METHODS The mouse tendon-bone reconstruction model was established, and the mice were randomly divided into 3 groups: the control group, the hydrogel group, and the hydrogel+exosome group, with 30 mice in each group. At 7 days, 14 days, and 1 month after surgery, tendon-bone junction samples were harvested, and the macrophage polarization and tendon-bone healing were evaluated based on histology, immunofluorescence, and quantitative RT-PCR (qRT-PCR) analysis. RESULTS In the early phase, we observed significantly higher numbers of M2 macrophages and more anti-inflammatory and chondrogenic-related factors in the hydrogel+BMSC-Exos group compared with the control group and the hydrogel group. The M1 macrophages and related proinflammatory factors decreased. Cell apoptosis decreased in the hydrogel+BMSC-Exos group, while cell proliferation increased; in particular, the CD146+ stem cells substantially increased. At 1 month after surgery, there was more fibrocartilage in the hydrogel+BMSC-Exos group than in the other groups. Biomechanical testing showed that the maximum force, strength, and elastic modulus were significantly improved in the hydrogel+BMSC-Exos group. CONCLUSIONS Our study provides evidence that the local administration of BMSC-Exos promotes the formation of fibrocartilage by increasing M2 macrophage polarization in tendon-to-bone healing, leading to improved biomechanical properties. These findings provide a basis for the potential clinical use of BMSC-Exos in tendon-bone repair.

Aspirin promotes tenogenic differentiation of tendon stem cells and facilitates tendinopathy healing through regulating the GDF7/Smad1/5 signaling pathway.

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries.

Absence of estrogen receptor beta leads to abnormal adipogenesis during early tendon healing by an up-regulation of PPARγ signalling.

Achilles tendon injury is one of the challenges of sports medicine, the aetiology of which remains unknown. For a long time, estrogen receptor ß (ERß) has been known as a regulating factor of the metabolism in many connective tissues, such as bone, muscle and cartilage, but little is known about its role in tendon. Recent studies have implicated ERß as involved in the process of tendon healing. Tendon-derived stem cells (TDSCs) are getting more and more attention in tendon physiological and pathological process. In this study, we investigated how ERß played a role in Achilles tendon healing. Achilles tendon injury model was established to analyse how ERß affected on healing process in vivo. Cell proliferation assay, Western blots, qRT-PCR and immunocytochemistry were performed to investigate the effect of ERß on TDSCs. Here, we showed that ERß deletion in mice resulted in inferior gross appearance, histological scores and, most importantly, increased accumulation of adipocytes during the early tendon healing which involved activation of peroxisome proliferator-activated receptor γ (PPARγ) signalling. Furthermore, in vitro results of ours confirmed that the abnormity might be the result of abnormal TDSC adipogenic differentiation which could be partially reversed by the treatment of ERß agonist LY3201. These data revealed a role of ERß in Achilles tendon healing for the first time, thereby providing a new target for clinical treatment of Achilles tendon injury.

[Research progress of structured repair of tendon-bone interface].

In sports system, the tendon-bone interface has the effect of tensile and bearing load, so the effect of healing plays a crucial role in restoring joint function. The process of repair is the formation of scar tissue, so it is difficult to achieve the ideal effect for morphology and biomechanical strength. The tissue engineering method can promote the tendon-bone interface healing from the seed cells, growth factors, and scaffolds, and is a new direction in the field of development of the tendon-bone interface healing.

Aspirin inhibits inflammation and scar formation in the injury tendon healing through regulating JNK/STAT-3 signalling pathway.

OBJECTIVELY: Tendinopathy is a common problem in sports medicine which can lead to severe morbidity. Aspirin, as the classical representative of non-steroidal anti-inflammatory drugs (NSAIDs) for its anti-inflammatory and analgesic actions, has been commonly used in treating tendinopathy. While its treatment effects on injury tendon healing are lacking, illuminating the underlying mechanism may provide scientific basis for clinical treatment. MATERIALS AND METHODS: Firstly, we used immunohistochemistry and qRT-PCR to detect changes in CD14, CD206, iNOS, IL-6, IL-10, MMP-3, TIMP-3, Col-1a1, biglycan, Comp, Fibronectin, TGF-ß1，ACAN，EGR-1 and FMOD. Next, Western blot was used to measure the protein levels (IL-6, IL-10, TGF-ß1, COMP, TIMP-3, STAT-3/P-STAT-3 and JNK/P-JNK) in TSCs. Then, migration and proliferation of TSCs were measured through wound healing test and BrdU staining. Finally, the mechanical properties of injury tendon were detected. RESULTS: After aspirin treatment, the inflammation and scar formation in injury tendon were significantly inhibited by aspirin. Still, tendon's ECM was positively balanced. Increasing migration and proliferation ability of TSCs induced by IL-1ß were significantly reversed. JNK/STAT-3 signalling pathway participated in the process above. In addition, biomechanical properties of injury tendon were significantly improved. CONCLUSIONS: Taken together, the findings suggested that aspirin inhibited inflammation and scar formation via regulation of JNK/STAT-3 signalling and decreased rerupture risk of injury tendon. Aspirin could be an ideal therapeutic strategy in tendon injury healing.

Exosomes from tendon stem cells promote injury tendon healing through balancing synthesis and degradation of the tendon extracellular matrix.

Tendon injuries are common musculoskeletal system disorders in clinical, but the regeneration ability of tendon is limited. Tendon stem cells (TSCs) have shown promising effect on tissue engineering and been used for the treatment of tendon injury. Exosomes that serve as genetic information carriers have been implicated in many diseases and physiological processes, but effect of exosomes from TSCs on tendon injury repair is unclear. The aim of this study is to make clear that the effect of exosomes from TSCs on tendon injury healing. Exosomes were harvested from conditioned culture media of TSCs by a sequential centrifugation process. Rat Achilles tendon tendinopathy model was established by collagenase-I injection. This was followed by intra-Achilles-tendon injection with TSCs or exosomes. Tendon healing and matrix degradation were evaluated by histology analysis and biomechanical test at the post-injury 5 weeks. In vitro, TSCs treated with interleukin 1 beta were added by conditioned medium including exosomes or not, or by exosomes or not. Tendon matrix related markers and tenogenesis related markers were measured by immunostaining and western blot. We found that TSCs injection and exosomes injection significantly decreased matrix metalloproteinases (MMP)-3 expression, increased expression of tissue inhibitor of metalloproteinase-3 (TIMP-3) and Col-1a1, and increased biomechanical properties of the ultimate stress and maximum loading. In vitro, conditioned medium with exosomes and exosomes also significantly decreased MMP-3, and increased expression of tenomodulin, Col-1a1 and TIMP-3. Exosomes from TSCs could be an ideal therapeutic strategy in tendon injury healing for its balancing tendon extracellular matrix and promoting the tenogenesis of TSCs.

High Concentration of Aspirin Induces Apoptosis in Rat Tendon Stem Cells via Inhibition of the Wnt/ß-Catenin Pathway.

BACKGROUND/AIMS: Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in clinical practice to relieve fever and pain. Aspirin, as a representative NSAID, has been widely used in the treatment of tendinopathy. Some reports have demonstrated that aspirin can induce apoptosis in cancer cells. However, evidence regarding aspirin treatment for tendinopathy, especially the effect of this treatment on tendon stem cells (TSCs), is lacking. Understanding the effect of aspirin on tendinopathy may provide a basis for the rational use of NSAIDs in clinical practice. The aim of our study was to determine whether aspirin induces apoptosis in rat TSCs via the Wnt/ß-catenin pathway. METHODS: First, we used flow cytometry and fluorescence to detect TSC apoptosis. Protein expression of the apoptosis-related caspase-3 pathway was investigated via western blot analysis. Next, we used western blotting to determine the effect of aspirin on the Wnt/ß-catenin pathway. We used immunostaining to detect the levels of Bcl2, cleaved caspase-3, and P-ß-catenin in the Achilles tendon. Finally, we used flow cytometry, fluorescence, and western blotting to investigate the aspirin-induced apoptosis of TSCs via the Wnt/ß-catenin pathway. RESULTS: Aspirin induced morphological apoptosis in rat TSCs via the mitochondrial/caspase-3 pathway and induced cellular apoptosis in the Achilles tendon. Apoptosis was partly reversed after adding the Wnt signaling activator Wnt3a and lithium chloride (LiCl, a GSK-3ß inhibitor). Aspirin administration led to a dose-dependent increase in COX-2 expression. Apoptosis was promoted after adding the COX-2 inhibitor NS398. CONCLUSION: The Wnt/ß-catenin pathway plays a vital role in aspirin-induced apoptosis by regulating mitochondrial/caspase-3 function. Elevating COX-2 levels may protect cells against apoptosis. More importantly, the results remind us to consider the apoptotic effect of aspirin on TSCs and tendon cells when aspirin is administered to treat tendinopathy. The relationship between the positive and negative effects of aspirin remains a subject for future study.

Initial stability and stress distribution of ankle arthroscopic arthrodesis with three kinds of 2-screw configuration fixation: a finite element analysis.

BACKGROUND: Arthroscopic ankle arthrodesis (AAA) is recognized as the standard treatment for the end-stage ankle arthritis. Two-screw configuration fixation is a typical technique for AAA; however, no consensus has been reached on how to select most suitable inserted position and direction. For better joint reduction, we developed a new configuration (2 home run-screw configuration: 2 screws are inserted from the lateral-posterior and medial-posterior malleolus into the talar neck) and investigated whether it turned out to be better than the other commonly used 2-screw configurations. METHODS: In this study, we investigated three kinds of 2-screw configurations: 2 "home run"-screw configuration (group A), crossed transverse configuration (the screw is inserted from the medial malleolus into the anterior talus and the other from the lateral tibia maintains posterior talus, group B), and 2 parallel screw configuration (2 parallel screws are inserted from the posteromedial side of the tibia into talus, group C). The effects of the above three insertions on the loading stress of the tibio-talar joint were comparatively analyzed with a three-dimensional finite element model. RESULTS: Group A was better than groups B and C in respect of stress distribution uniformity and superior to both groups B and C in anti-flexion strength and anti-internal rotation strength. Group A was slightly worse than group C but better than group B in anti-dorsiflexion and anti-valgus and varus strength. CONCLUSIONS: Two "home run"-screw configuration facilitates the reduction of anterior talus dislocation of end-stage ankle arthritis. Our finite element analysis demonstrates the configuration is superior to crossed transverse and parallel configuration for arthroscopic ankle arthrodesis in terms of stress distribution and initial stability.

Interleukin-15 facilitates muscle regeneration through modulation of fibro/adipogenic progenitors.

BACKGROUND: Chronic muscle injury is characteristics of fatty infiltration and fibrosis. Recently, fibro/adipogenic progenitors (FAPs) were found to be indispensable for muscular regeneration while were also responsible for fibrosis and fatty infiltration in muscle injury. Many myokines have been proven to regulate the adipose or cell proliferation. Because the fate of FAPs is largely dependent on microenvironment and the regulation of myokines on FAPs is still unclear. We screened the potential myokines and found Interleukin-15 (IL-15) may regulate the fatty infiltration in muscle injury. In this study, we investigated how IL-15 regulated FAPs in muscle injury and the effect on muscle regeneration. METHODS: Cell proliferation assay, western blots, qRT-PCR, immunohistochemistry, flow cytometric analysis were performed to investigate the effect of IL-15 on proliferation and adipogensis of FAPs. Acute muscle injury was induced by injection of glycerol or cardiotoxin to analyze how IL-15 effected on FAPs in vivo and its function on fatty infiltration or muscle regeneration. RESULTS: We identified that the expression of IL-15 in injured muscle was negatively associated with fatty infiltration. IL-15 can stimulate the proliferation of FAPs and prevent the adipogenesis of FAPs in vitro and in vivo. The growth of FAPs caused by IL-15 was mediated through JAK-STAT pathway. In addition, desert hedgehog pathway may participate in IL-15 inhibiting adipogenesis of FAPs. Our study showed IL-15 can cause the fibrosis after muscle damage and promote the myofiber regeneration. Finally, the expression of IL-15 was positively associated with severity of fibrosis and number of FAPs in patients with chronic rotator cuff tear. CONCLUSIONS: These findings supported the potential role of IL-15 as a modulator on fate of FAPs in injured muscle and as a novel therapy for chronic muscle injury.

[Imaging evaluation on adaptability of proximal humeral anatomy after shoulder replacement with individualized shoulder prosthesis].

OBJECTIVE: Modern shoulder prosthesis has evolved through four generations. And the fourth generation technology has a core three-dimensional design of restoring 3D reconstruction of proximal humeral anatomy. Thus a new shoulder prosthesis is developed on the basis of the technology of 3D prosthesis. Assessment of whether shoulder prosthesis can restore individualized reconstruction of proximal humeral anatomy is based on the adaptability of proximal humeral anatomy. To evaluate the adaptability of proximal humeral anatomy through measuring the parameters of proximal humeral anatomy after shoulder replacement with individualized shoulder prosthesis and compare with normal data. METHODS: The parameters of proximal humeral anatomy were analyzed and evaluated for a total of 12 cases undergoing shoulder replacement with individualized shoulder prosthesis. The relevant anatomical parameters included neck-shaft angle (NSA), retroversion angle (RA), humeral head height (HH) and humeral head diameter (HD). And the anatomical parameters were compared with the data from normal side. RESULTS: All underwent shoulder replacement with individualized shoulder prosthesis. The postoperative parameters of proximal humeral anatomy were compared with those of normal side. And the difference of NSA was < 3°, RA < 3°, HH < 3 mm and HD < 2 mm. And paired-sample t test was used to study the parameters of proximal humeral anatomy between postoperative and normal side. The normal and postoperative NSA was (140.2 ± 6.8)° and (139.5 ± 6.6)° respectively, RA (34.4 ± 3.3)° and (33.8-3.1)°, HH (15.3 ± 2.1) mm and (14.6+0.9) mm, HW (42.2 ± 2.82) mm and (41.8 ± 2.33) mm respectively. No significant difference existed between two groups (P > 0.05). CONCLUSION: Individualized shoulder prosthesis has excellent adaptability to shoulder. All core parameters are freely adjustable and specification models may be optimized. With matching tools, individualized shoulder prosthesis improves the accuracy and reliability in shoulder replacement.