Tendon stem cells seeded on dynamic chondroitin sulfate and chitosan hydrogel scaffold with BMP2 enhance tendon-to-bone healing.

Failure to adequately reconstruct the tendon-to-bone interface constitutes the primary etiology underlying rotator cuff retear after surgery. The purpose of this study is to construct a dynamic chondroitin sulfate and chitosan hydrogel scaffold (CHS) with bone morphogenetic protein 2 (BMP2), then seed tendon stem cells (TSCs) on BMP2-CHS for the rotator cuff reconstruction of tendon-to-bone interface. In this dynamic hydrogel system, the scaffold could not only have good biocompatibility and degradability but also significantly promote the proliferation and differentiation of TSCs. The ability of BMP2-CHS combined with TSCs to promote regeneration of tendon-to-bone interface was further verified in the rabbit rotator cuff tear model. The results showed that BMP2-CHS combined with TSCs could induce considerable collagen, fibrocartilage, and bone arrangement and growth at the tendon-to-bone interface and promote the biomechanical properties. Overall, TSCs seeded on CHS with BMP2 can enhance tendon-to-bone healing and provide a new possibility for improving the poor prognosis of rotator cuff surgery.

Small extracellular vesicles derived from tendon stem cells promote the healing of injured Achilles tendons by regulating miR-145-3p.

The therapeutic role of tendon stem cells (TSCs) in tendon-related injuries has been well documented. Small extracellular vesicles (sEVs) are being increasingly used as new biotherapeutic agents for various diseases. Therefore, the potential function of TSC-sEVs in tendon injury repair warrants further investigation. In this study, we explored the effects of TSC-sEVs on TSC proliferation, migration, and differentiation in vitro in an autocrine manner. We further used a novel exosomal topical treatment with TSC-sEVs loaded with gelatin methacryloyl (GelMA) hydrogel in vivo; we mixed sufficient amounts of TSC-sEVs with GelMA hydrogel to cover the damaged molded Achilles tendon tissue and then exposed them to UV irradiation for coagulation. GelMA loading ensured that TSC-sEVs were slowly released at the injury site over a long period, thereby achieving their full local therapeutic effects. Treatment with TSC-sEVs loaded with GelMA significantly improved the histological score of the regenerated tendon by increasing the tendon expression while inhibiting the formation of excessive ossification and improving the mechanical properties of the tissue. Moreover, miRNA sequencing in TSC-sEVs, TSCs, and TSCs receiving sEVs revealed that TSC-sEVs altered the miRNA expression profile of TSCs, with increased expression of miR-145-3p. In conclusion, our study demonstrates that TSC-sEVs can play a key role in treating tendon injuries and that loading them with GelMA can enhance their effect in vivo. Moreover, miR-145-3p has a major functional role in the effect of TSC-sEVs. This study offers new therapeutic ideas for the local treatment of Achilles tendon injuries using sEVs. STATEMENT OF SIGNIFICANCE: In this study, we demonstrated that TSC-sEVs play a key role in treating tendon injuries and that loading them with GelMA hydrogel can act as a fixation and slow release in vivo. Moreover, it identifies the major functional role of miR-145-3p in the effect of TSCs that were identified and validated by miRNA sequencing. Our study provides a basis for further research on GelMA slow-release assays that have potential clinical applications. It offers new therapeutic ideas for the local treatment of Achilles tendon injuries using TSC-sEVs.

The regulation of tendon stem cell distribution, morphology, and gene expression by the modulus of microfibers.

The mechanical properties of a stem cell culture substrate significantly impact cell adhesion, survival, migration, proliferation, and differentiation in vitro. A major challenge in engineering artificial stem cell substrate is to properly identify the relevant physical features of native stem cell niches, which are likely different for each stem cell type. The behavior of tendon stem cells has potentially significant implications for tendon repair. Here, microfiber scaffolds with various modulus of elasticity are fabricated by near-field electrospinning, and their regulating effects on the in vitro behavior of tendon stem cells (TSCs) are discussed in this study. The number of pseudopodia shows a biphasic relationship with the modulus of scaffold. The proliferation, polarization ratio and alignment degree along the fibers of the TSCs increase with the increase of fiber modulus. TSCs cultured on the scaffold with moderate modulus (1429 MPa) show the upregulation of tendon-specific genes (Col-I, Tnmd, SCX and TNCF). These microfiber scaffolds provide great opportunities to modulate TSCs behavior at the micrometer scales. In conclusion, this study provides an instructive mechanical microenvironment for TSCs behaviors and may lead to the development of desirable engineered artificial stem cell substrate for tendon healing.

CD200 as a Potential New Player in Inflammation during Rotator Cuff Tendon Injury/Repair: An In Vitro Model.

Rotator cuff tendon (RCT) disease results from multifactorial mechanisms, in which inflammation plays a key role. Pro-inflammatory cytokines and tendon stem cell/progenitor cells (TSPCs) have been shown to participate in the inflammatory response. However, the underlying molecular mechanism is still not clear. In this study, flow cytometry analyses of different subpopulations of RCT-derived TSPCs demonstrate that after three days of administration, TNFα alone or in combination with IFNγ significantly decreases the percentage of CD146+CD49d+ and CD146+CD49f+ but not CD146+CD109+ TSPCs populations. In parallel, the same pro-inflammatory cytokines upregulate the expression of CD200 in the CD146+ TSPCs population. Additionally, the TNFα/IFNγ combination modulates the protein expression of STAT1, STAT3, and MMP9, but not fibromodulin. At the gene level, IRF1, CAAT (CAAT/EBPbeta), and DOK2 but not NF-κb, TGRF2 (TGFBR2), and RAS-GAP are modulated. In conclusion, although our study has several important limitations, the results highlight a new potential role of CD200 in regulating inflammation during tendon injuries. In addition, the genes analyzed here might be new potential players in the inflammatory response of TSPCs.

TGFß1-transfected tendon stem cells promote tendon fibrosis.

BACKGROUND: In aged people, tendon injuries frequently occur during sporting and daily activities. In clinical practice, typical physiotherapeutic, pharmacotherapeutic, and surgical techniques do not result in the full recovery of injured tendons, which may lead to chronic degenerative disease. METHODS: We first isolated tendon stem cells (TSCs) from rats and transfected them with the TGFß1 gene, resulting in TGFß1-TSCs. The proliferation of TSCs was detected using the Cell Counting Kit 8, and TSCs were identified by immunofluorescence analysis and differentiation capacity analysis. Aggrecan, COL2A1, alpha smooth muscle actin (α-SMA), and p-Smad2 expression levels were detected using western blotting and quantitative reverse transcription polymerase chain reaction. Additionally, a tendon injury model was generated to explore the effect of TGFß1 on the repair of the tendon by TSCs. RESULTS: Compared with fibrinogen treatment, TSC + fibrinogen or TGFß1-TSC + fibrinogen treatment significantly promoted the fibrosis of injured tendons, as evidenced by histological analyses, with TGFß1-TSC + fibrinogen having a greater effect than TSC + fibrinogen. In TGFß1-TSCs, increased expression levels of aggrecan and COL2A1 indicated that TGFß1 signaling induced chondrogenic differentiation. Meanwhile, the increased collagen and α-SMA protein levels indicated that TGFß1 promoted fibrogenesis. Additionally, TGFß1 stimulated the production of phosphorylated Smad2 in TSCs, which suggested that the chondrogenic and fibrogenic differentiation of TSCs, as well as tissue regeneration, may be associated with the TGFß1/Smad2 pathway. CONCLUSION: TGFß1-TSC therapy may be a candidate for effective tendon fibrosis.

Stem Cells in Tendon Regeneration and Factors governing Tenogenesis.

Tendons are connective tissue structures of paramount importance to the human ability of locomotion. Tendinopathy and tendon rupture can be resistant to treatment and often recurs, thus resulting in a significant health problem with a relevant social impact worldwide. Unfortunately, existing treatment approaches are suboptimal. A better understanding of the basic biology of tendons may provide a better way to solve these problems and promote tendon regeneration. Stem cells, either obtained from tendons or non-tendon sources, such as bone marrow (BMSCs), adipose tissue (AMSCs), as well as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have received increasing attention toward enhancing tendon healing. There are many studies showing that stem cells can contribute to improving tendon healing. Hence, in this review, the current knowledge of BMSCs, AMSCs, TSPCs, ESCs, and iPSCs for tendon regeneration, as well as the advantages and limitations among them, has been highlighted. Moreover, the transcriptional and bioactive factors governing tendon healing processes have been discussed.

Effect of Eriocitrin on Cell Proliferation, Apoptosis, Migration, and Scar Formation-Related Genes Expression in Tendon Stem Cells.

Tendinopathy is a common disease in elite and recreational athletes, and Eriocitrin is a flavonoid compound, which has antioxidant properties. The present study was undertaken to investigate the effect of Eriocitrin on tendon stem cells in vitro. Different concentrations of Eriocitrin (0, 25, 50, 75 µM) were used to treat tendon stem cells, cell proliferation was determined by CCK8 assay, apoptosis was detected by propidium iodide (PI) staining assays and caspase-3 activity assessment, wound-healing assaywas selected to detect the migration, and RT-PCR was chosen to detect the expression levels of scar formation-related gene markers (COMP, Fibronectin, and Biglycan). Eriocitrin treatment promoted cell proliferation of tendon stem cells in dose-dependent manner, and it reduced the apoptotic activities and improved the migration of tendon stem cells. It inhibited COMP, Biglycan, and Fibronectin expression. In summary, eriocitrin promoted cell proliferation of tendon stem cells, improved the migration of tendon stem cells, and inhibited the expression levels of some scar formation-related gene markers.

Potential Mechanisms of the Impact of Hepatocyte Growth Factor Gene-Modified Tendon Stem Cells on Tendon Healing.

The therapeutic impact of stem cells is potentially largely attributable to secretion of exosomes and soluble factors. The present study evaluates the impact of hepatocyte growth factor (HGF)-expressing tendon stem cells (TSCs) on tendon healing in a rat model. Patellar tendon TSCs were isolated and underwent transfection with lentiviral vectors containing HGF or green fluorescent protein (GFP) genes. In vivo, immunohistochemistry of tendons sampled 1 week postsurgery demonstrated that all stem cell-treated groups exhibited higher numbers of CD163+ M2 monocytes and IL-10+ cells (anti-inflammatory), and lower numbers of CCR7+ M1 monocytes and IL-6+ as well as COX-2+ cells (pro-inflammatory). Effects were most pronounced in the HGF-expressing TSCs (TSCs + HGF) treated group. Histology ± immunohistochemistry of tendons sampled 4 and 8 weeks postsurgery demonstrated that all stem cell-treated groups exhibited more ordered collagen fiber arrangement and lower levels of COLIII, α-SMA, TGF-ß1, and fibronectin (proteins relevant to fibroscarring). Effects were most pronounced in the TSCs + HGF-treated group. For the in vitro study, isolated tendon fibroblasts pretreated with TGF-ß1 to mimic the in vivo microenvironment of tendon injury were indirectly cocultured with TSCs, TSCs + GFP, or TSCs + HGF using a transwell system. Western blotting demonstrated that all stem cell types decreased TGF-ß1-induced increases in fibroblast levels of COX-2, COLIII, and α-SMA, concomitant with decreased activation of major TGF-ß1 signaling pathways (p38 MAPK, ERK1/2, but not Smad2/3). This effect was most pronounced for TSCs + HGF, which also decreased the TGF-ß1-induced increase in activation of the Smad2/3 signaling pathway. The presence of specific inhibitors of these pathways during fibroblast TGF-ß1 stimulation also attenuated increases in levels of COX-2, COLIII, and α-SMA. In conclusion, TSCs + HGF, which exhibit HGF overexpression, may promoting tendon healing via decreasing inflammation and fibrosis, perhaps partly via inhibiting TGF-ß1-induced signaling. These findings identify a novel potential therapeutic strategy for tendon injuries, warranting additional research.

Mechanical Overloading Induced-Activation of mTOR Signaling in Tendon Stem/Progenitor Cells Contributes to Tendinopathy Development.

Despite the importance of mechanical loading in tendon homeostasis and pathophysiology, the molecular responses involved in the mechanotransduction in tendon cells remain unclear. In this study, we found that in vitro mechanical loading activated the mammalian target of rapamycin (mTOR) in rat patellar tendon stem/progenitor cells (TSCs) in a stretching magnitude-dependent manner. Application of rapamycin, a specific inhibitor of mTOR, attenuated the phosphorylation of S6 and 4E-BP1 and as such, largely inhibited the mechanical activation of mTOR. Moreover, rapamycin significantly decreased the proliferation and non-tenocyte differentiation of PTSCs as indicated by the reduced expression levels of LPL, PPARγ, SOX-9, collagen II, Runx-2, and osteocalcin genes. In the animal studies, mice subjected to intensive treadmill running (ITR) developed tendon degeneration, as evidenced by the formation of round-shaped cells, accumulation of proteoglycans, and expression of SOX-9 and collagen II proteins. However, daily injections of rapamycin in ITR mice reduced all these tendon degenerative changes. Collectively, these findings suggest that mechanical loading activates the mTOR signaling in TSCs, and rapamycin may be used to prevent tendinopathy development by blocking non-tenocyte differentiation due to mechanical over-activation of mTOR in TSCs.

Adipose-derived mesenchymal stromal cell-derived exosomes promote tendon healing by activating both SMAD1/5/9 and SMAD2/3.

BACKGROUND: The use of adipose-derived mesenchymal stromal cell-derived exosomes (ADSC-Exos) may become a new therapeutic method in biomedicine owing to their important role in regenerative medicine. However, the role of ADSC-Exos in tendon repair has not yet been evaluated. Therefore, we aimed to clarify the healing effects of ADSC-Exos on tendon injury. METHODS: The adipose-derived mesenchymal stromal cells (ADSCs) and tendon stem cells (TSCs) were isolated from the subcutaneous fat and tendon tissues of Sprague-Dawley rats, respectively, and exosomes were isolated from ADSCs. The proliferation and migration of TSCs induced by ADSC-Exos were analyzed by EdU, cell scratch, and transwell assays. We used western blot to analyze the tenogenic differentiation of TSCs and the role of the SMAD signaling pathways. Then, we explored a new treatment method for tendon injury, combining exosome therapy with local targeting using a biohydrogel. Immunofluorescence and immunohistochemistry were used to detect the expression of inflammatory and tenogenic differentiation after tendon injury, respectively. The quality of tendon healing was evaluated by hematoxylin-eosin (H&E) staining and biomechanical testing. RESULTS: ADSC-Exos could be absorbed by TSCs and promoted the proliferation, migration, and tenogenic differentiation of these cells. This effect may have depended on the activation of the SMAD2/3 and SMAD1/5/9 pathways. Furthermore, ADSC-Exos inhibited the early inflammatory reaction and promoted tendon healing in vivo. CONCLUSIONS: Overall, we demonstrated that ADSC-Exos contributed to tendon regeneration and provided proof of concept of a new approach for treating tendon injuries.

Exosomes derived from tendon stem cells promote cell proliferation and migration through the TGF ß signal pathway.

Tendon stem cells (TSCs) are a kind of progenitor cells found in tendon niches, which play a key role in the repair of tendon injuries. Exosomes that mediate cell communication are involved in physiological processes and various diseases, while the effect of exosomes derived from TSCs (TSC-exo) on TSCs is still unclear. The purpose of this study is to explore the effect of TSC-exo on TSCs. Analyzing the characteristics of TSC-exo, we found that the TSC-exo were enriched in a large amount of transforming growth factor ß (TGF ß) by western blotting. We also found that the TGF ß carried by TSC-exo can effectively accelerate the proliferation and migration of TSCs. We further found that TGF ß carried by TSC-exo can activate the TGF ß-Smad2/3 and the ERK1/2 signaling pathway in TSCs. Furthermore, matrix metalloenzyme 2 (MMP2), a downstream molecule of Smad2, is regulated by TGF ß carried by TSC-exo. Collectively, our findings provide molecular insights into TSC-exo and indicate that TSC-exo are a potential strategy for treating tendon injuries.

Ascorbic acid mitigates the deleterious effects of nicotine on tendon stem cells.

Purpose: Nicotine causes tendon degeneration, whereas ascorbic acid imparts beneficial effects on tendon cells. Tendon stem cells (TSCs) play a vital role in maintaining tissue integrity and promoting restoration of structure and function after tendon injury. In the present study, cell culture experiments were performed to determine the effects of nicotine on TSCs and whether ascorbic acid supplementation could antagonize the action of high concentration nicotine. Methods: After treatment with nicotine and ascorbic acid, TSC proliferation, migration, stemness, apoptosis, and differentiation were analyzed. Results: TSC proliferation and expression of stem cell markers were significantly impaired by a high concentration of nicotine (1000 ng/mL), but a lower concentration (100 ng/mL) induced proliferative effects in TSCs. Moreover, the highest concentration of nicotine tested (1000 ng/mL) significantly inhibited the migratory ability of TSCs, while relatively high concentrations (100 and 1000 ng/mL) significantly (p < 0.05) up-regulated non-tenocyte genes. When ascorbic acid was added, the inhibitory effects of nicotine on the proliferation, migration, and stemness of TSCs were reversed. In addition, flow cytometry analysis showed that these nicotine concentrations could induce cell apoptosis, while the addition of ascorbic acid inhibited apoptosis. Conclusion: Addition of ascorbic acid partially reversed the inhibitory effect of a high concentration of nicotine. These findings indicate that while nicotine impairs the biological characteristics of TSCs, ascorbic acid can mitigate these deleterious effects and, therefore, may be useful for decreasing nicotine-induced tendon degeneration.

Effect of cytokines and platelet-rich plasma on tendon derived stem cells / 中国组织工程研究

BACKGROUND: Tendon derived stem cells exist in the tendon and have unique functions for tendon repair. Different cytokines have different effects on the proliferation and differentiation of tendon derived stem cells. Platelet-rich plasma refers to the blood product obtained from the whole blood through gradient centrifugation and stratification, which contains a variety of cytokines that could help to promote the regeneration of ligaments and tendons. OBJECTIVE: To investigate the latest progress of effects of cytokines and platelet-rich plasma on proliferation and differentiation of tendon derived stem cells. METHODS: Using “tendon derived stem cells, tendon stem/progenitor cells, tendon stem cell, platelet-rich plasma, ligament injury” as keywords in English and “tendon derived stem cells, platelet-rich plasma, ligament injury” in Chinese, the first author searched PubMed, CNKI, and Wanfang for relevant articles published from 2007 to 2019. Literature unrelated to the purpose of the study and repetitive literature were excluded, and 83 articles that meet the criteria were included for review. RESULTS AND CONCLUSION: Tendon derived stem cells are ideal cells for the treatment of tendon injury in cell transplantation. Its proliferation and differentiation are influenced by cytokines. Platelet-rich plasma contains a large number of cytokines, which can stimulate the proliferation and differentiation of tendon derived stem cells and have the potential to become a carrier of cell transplantation. Exploring the relationship between cytokines and proliferation and differentiation of tendon stem cells will provide a new approach for the clinical application of tendon derived stem cells.

Tendon stem cell-derived exosomes regulate inflammation and promote the high-quality healing of injured tendon.

BACKGROUND: Tendon stem cells (TSCs) have been reported to hold promises for tendon repair and regeneration. However, less is known about the effects of exosomes derived from TSCs. Therefore, we aimed to clarify the healing effects of TSC-derived exosomes (TSC-Exos) on tendon injury. METHODS: The Achilles tendons of Sprague-Dawley male rats were used for primary culture of TSCs and tenocytes, and exosomes were isolated from TSCs. The proliferation of tenocytes induced by TSC-Exos was analyzed using an EdU assay; cell migration was measured by cell scratch and transwell assays. We used western blot to analyze the role of the PI3K/AKT and MAPK/ERK1/2 signaling pathways. In vivo, Achilles tendon injury models were created in Sprague-Dawley rats. Rats (n = 54) were then randomly assigned to three groups: the TSC-Exos group, the GelMA group, and the control group. We used immunofluorescence to detect changes in the expression of inflammatory and apoptotic markers at 1 week after surgery. Histology and changes in expression of extracellular matrix (ECM)-related indices were assessed by hematoxylin-eosin (H&E) staining and immunohistochemistry at 2 and 8 weeks. The collagen fiber diameter of the healing tendon was analyzed at 8 weeks by transmission electron microscopy (TEM). RESULTS: TSC-Exos were taken up by tenocytes, which promoted the proliferation and migration of cells in a dose-dependent manner; this process may depend on the activation of the PI3K/AKT and MAPK/ERK1/2 signaling pathways. At 1 week after surgery, we found that inflammation and apoptosis were significantly suppressed by TSC-Exos. At 2 and 8 weeks, tendons treated with TSC-Exos showed more continuous and regular arrangement in contrast to disorganized tendons in the GelMA and control groups, and TSC-Exos may help regulate ECM balance and inhibited scar formation. Further, at 8 weeks, the TSC-Exos group had a larger diameter of collagen compared to the control group. CONCLUSIONS: Our data suggest that TSC-Exos could promote high-quality healing of injured tendon, which may be a promising therapeutic approach for tendon injury.

Nudt21-mediated alternative polyadenylation of HMGA2 3'-UTR impairs stemness of human tendon stem cell.

Tendon-derived stem cells (TSCs) play a primary role in tendon physiology, pathology, as well as tendon repair and regeneration after injury. TSCs are often exposed to mechanical loading-related cellular stresses such as oxidative stress, resulting in loss of stemness and multipotent differentiation potential. Cytoprotective autophagy has previously been identified as an important mechanism to protect human TSCs (hTSCs) from oxidative stress induced impairments. In this study, we found that high-mobility AT-hook 2 (HMGA2) overexpression protects hTSCs against H2O2-induced loss of stemness through autophagy activation. Evidentially, H2O2 treatment increases the expression of Nudt21, a protein critical to polyadenylation site selection in alternative polyadenylation (APA) of mRNA transcripts. This leads to increased cleavage and polyadenylation of HMGA2 3'-UTR at the distal site, resulting in increased HMGA2 silencing by the microRNA let-7 and reduced HMGA2 expression. In conclusion, Nudt21-regulated APA of HMGA2 3'-UTR and subsequent HMGA2 downregulation mediates oxidative stress induced hTSC impairments.

Aspirin inhibits adipogenesis of tendon stem cells and lipids accumulation in rat injury tendon through regulating PTEN/PI3K/AKT signalling.

Tendon injury repairs are big challenges in sports medicine, and fatty infiltration after tendon injury is very common and hampers tendon injury healing process. Tendon stem cells (TSCs), as precursors of tendon cells, have shown promising effect on injury tendon repair for their tenogenesis and tendon extracellular matrix formation. Adipocytes and lipids accumulation is a landmark event in pathological process of tendon injury, and this may induce tendon rupture in clinical practice. Based on this, it is important to inhibit TSCs adipogenesis and lipids infiltration to restore structure and function of injury tendon. Aspirin, as the representative of non-steroidal anti-inflammatory drugs (NSAIDs), has been widely used in tendon injury for its anti-inflammatory and analgesic actions, but effect of aspirin on TSCs adipogenesis and fatty infiltration is still unclear. Under adipogenesis conditions, TSCs were treated with concentration gradient of aspirin. Oil red O staining was performed to observe changes of lipids accumulation. Next, we used RNA sequencing to compare profile changes of gene expression between induction group and aspirin-treated group. Then, we verified the effect of filtrated signalling on TSCs adipogenesis. At last, we established rat tendon injury model and compared changes of biomechanical properties after aspirin treatment. The results showed that aspirin decreased lipids accumulation in injury tendon and inhibited TSCs adipogenesis. RNA sequencing filtrated PTEN/PI3K/AKT signalling as our target. After adding the signalling activators of VO-Ohpic and IGF-1, inhibited adipogenesis of TSCs was reversed. Still, aspirin promoted maximum loading, ultimate stress and breaking elongation of injury tendon. In conclusion, by down-regulating PTEN/PI3K/AKT signalling, aspirin inhibited adipogenesis of TSCs and fatty infiltration in injury tendon, promoted biomechanical properties and decreased rupture risk of injury tendon. All these provided new therapeutic potential and medicine evidence of aspirin in treating tendon injury and tendinopathy.

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.

Extracellular vesicles from bone marrow-derived multipotent mesenchymal stromal cells regulate inflammation and enhance tendon healing.

BACKGROUND: Extracellular vesicles from bone marrow-derived multipotent mesenchymal stromal cells (BMSC-EVs) can play important roles in the repair of injured tissues. However, no reports have investigated the role and underlying mechanisms of BMSCs-EVs in the tendon repair process. We hypothesized that BMSC-EVs may play a role in modulating inflammation during tendon healing and improving tendon repair in a rat model of patellar tendon injury. METHODS: First, we created window defects in the patellar tendons of Sprague-Dawley rats. Rats (n = 16) were then randomly assigned to three groups: BMSC-EVs group, Fibrin group, and control group. Rats in the BMSC-EVs group were treated with BMSC-EVs and fibrin glue (25 µg in 10 µL). Rats in the fibrin group were treated with fibrin only, and those in the control group received no treatment. Histopathology, immunohistochemistry, and gene expression analyses were performed at 2 and 4 weeks after surgery. RESULTS: At 4 weeks, tendons treated with BMSC-EVs showed regularly aligned and compact collagen fibers as compared with the disrupted scar-like healing in rats in the fibrin and control groups. The expression of genes related to tendon matrix formation and tenogenic differentiation: collagen (COL)-1a1, scleraxis (SCX), and tenomodulin (TNMD) was significantly higher in the BMSC-EVs group than in the other two groups. With histopathology, we observed significantly higher numbers of CD146+ tendon stem cells and fewer numbers of apoptotic cells and C-C chemokine receptor type 7 (CCR7)-positive proinflammatory macrophages in the BMSC-EVs group. BMSC-EVs treatment also led to an increase in the expression of anti-inflammatory mediators (IL-10 and IL-4) at 2 weeks after surgery. CONCLUSIONS: Overall, our findings show that the local administration of BMSC-EVs promotes tendon healing by suppressing inflammation and apoptotic cell accumulation and increasing the proportion of tendon-resident stem/progenitor cells. These findings provide a basis for the potential clinical use of BMSC-EVs in tendon repair.

Co-cultured Bone-marrow Derived and Tendon Stem Cells: Novel Seed Cells for Bone Regeneration.

Tendon-bone healing after injury is an unsolved problem. Several types of stem cells are used as seed cells. However, the optimal co-culture ratio of different types of cells suitable for tissue engineering as well as the stimulator for facilitating the differentiation of stem cells in tendon-bone healing is unclear. In this study, the proliferation of both bone marrow-derived stem cells (BMSCs) and tendon stem cells (TSCs) was increased at a 1:1 co-cultured ratio, and proliferation was suppressed by Tenascin C (TNC). TNC treatment can promote osteogenesis or chondrogenesis of both BMSCs and TSCs under a 1:1 co-cultured ratio. In addition, the expression level of Rho-associated kinase (ROCK) increased in the process of TNC-induced osteogenesis and decreased in the process of TNC-induced chondrogenesis. Furthermore, the level of insulin-like growth factor 1 receptor (IGF-1R) and mitogen-activated protein kinase (MEK) was upregulated during the osteogenesis and chondrogenesis of both BMSCs and TSCs after TNC treatment. Although our study was conducted in rats with no direct evaluation of the resulting cells for tendon-bone healing and regeneration, we show that the proliferation of BMSCs and TSCs was enhanced under a 1:1 co-cultured ratio. TNC has a significant impact on the proliferation and differentiation of co-cultured BMSCs and TSCs. IGF-IR, ROCK, and MEK may become involved in the process after TNC treatment.