As a common complication of tendon injury, tendon adhesion is an unresolved problem in clinical work. The aim of this study was to investigate whether human umbilical cord mesenchymal stem cell-derived exosomes (HUMSC-Exos), one of the most promising new-generation cell-free therapeutic agents, can improve tendon adhesion and explore potential-related mechanisms. Methods: The rat Achilles tendon injury adhesion model was constructed in vivo, and the localization of HUMSC-Exos was used to evaluate the tendon adhesion. Rat fibroblast cell lines were treated with transforming growth factor β1 (TGF-β1) and/or HUMSC-Exos in vitro, and cell proliferation, apoptosis and gene expression were measured. MicroRNA (miRNA) sequencing and quantitative PCR (qPCR) analysis confirmed differential miRNAs. A specific miRNA antagonist (antagomir-21a-5p) was used to transform HUMSC-Exos and obtain modified exosomes to verify its efficacy and related mechanism of action. Results: In this study, we found HUMSC-Exos reduced rat fibroblast proliferation and inhibited the expression of fibrosis genes: collagen III (COL III) and α-smooth muscle actin (α-SMA) in vitro. In the rat tendon adhesion model, topical application of HUMSC-Exos contributed to relief of tendon adhesion. Specifically, the fibrosis and inflammationrelated genes were simultaneously inhibited by HUMSC-Exos. Further, miRNA sequencing of HUMSCs and HUMSC-Exos showed that miR-21a-3p was expressed at low abundance in HUMSC-Exos. The antagonist targeting miR-21a-3p was recruited for treatment of HUMSCs, and harvested HUMSC-Exos, which expressed low levels of miR-21a-3p, and expanded the inhibition of tendon adhesion in subsequent in vitro experiments. Conclusion: Our results indicate that HUMSC-Exos may manipulate p65 activity by delivering low-abundance miR-21a-3p, ultimately inhibiting tendon adhesion. The findings may be promising for dealing with tendon adhesion.

Background Exosomes are extracellular vesicles of nano-structures and represent an emerging nano-scale acellular therapy in recent years. Tendon regeneration is a sophisticated process in the field of microsurgery due to its poor natural healing ability. To date, no successful long-term solution has been provided for the healing of tendon injuries. Functional recovery requires advanced treatment strategies. Human umbilical cord mesenchymal stem cell-derived exosomes (HUMSC-Exos) are considered as promising cell-free therapeutic agents. However, few studies reported their potential in the tendon repair previously. In this study, we explored the roles and underlying mechanisms of HUMSC-Exos in the tendon regeneration. Results Expression of tendon‐specific markers in, and collagen deposition by, tendon-derived stem cells (TDSCs) treated with HUMSC-Exos increased in vitro. In a rat Achilles tendon injury model, treatment with HUMSC-Exos improved the histological structure, enhanced tendon-specific matrix components, and optimized biomechanical properties of the Achilles tendon. Findings in miRNA sequencing indicated a significant increase in miR-29a-3p in HUMSC-Exo-treated Achilles tendons. Next, luciferase assay in combination with western blot identified phosphatase and tensin homolog (PTEN) as the specific target of miR-29a-3p. Furthermore, we applied a miR-29a-3p-specific agonist to engineer HUMSC-Exos. These HUMSC-Exos overexpressing miR-29a-3p amplified the gain effects of HUMSC-Exos on tendon healing in vivo. To explore the underlying mechanisms, a transforming growth factor-β1 (TGF-β1) inhibitor (SB-431542), mTOR inhibitor (rapamycin), and engineered HUMSC-Exos were employed. The results showed that TGF-β1 and mTOR signaling were involved in the beneficial effects of HUMSC-Exos on tendon regeneration. Conclusion The findings in our study suggest that PTEN/mTOR/TGF-β1 signaling cascades may be a potential pathway for HUMSC-Exos to deliver miR-29a-3p for tendon healing and implicate a novel therapeutic strategy for tendon regeneration via engineered stem cell-derived exosomes. Graphic abstract

Traumatic peritendinous fibrosis is a worldwide clinical problem resulting in severe limb disability. Hydroxycamptothecin (HCPT) is an anti-neoplastic drug widely exploited in clinical practice. It has shown potential of anti-fibrosis in recent years. We previously demonstrated that HCPT inhibited the characterization of fibrosis in vitro. However, it is still unclear whether it ameliorates peritendinous adhesion in an in vivo animal tendon injury model. The underlying mechanism is also worth investigating. The present study aims to determine whether HCPT inhibits tendon adhesion and to explore the underlying mechanisms. In a rat tendon injury model, we observed that topical application of HCPT significantly attenuated peritendinous adhesion as revealed by the results of macroscopic observation, biomechanical, histological, immunohistochemical evaluation, western blot, and quantitative PCR (q-PCR) analyses. Furthermore, western blot and q-PCR analyses revealed that this phenomenon is correlated with HCPT activation of endoplasmic reticulum (ER) stress. In addition, in vitro studies show that HCPT significantly inhibits fibroblast proliferation and induces apoptosis by reducing the expression of extracellular matrix (ECM) proteins COL3A1 and α-smooth muscle actin (α-SMA). Finally, we employed small interfering RNA (siRNA) to target inositol requiring kinase 1 (IRE1) and activated transcription factor 6 (ATF-6) to verify that the effect of inhibitory fibrosis of HCPT disappears after knockdown of ATF-6 and IRE1, thereby suggesting that an anti-fibrotic effect of HCPT is mediated by the ER-dependent apoptotic pathway. In conclusion, our results indicate that HCPT inhibits peritendinous fibrosis through the ER-dependent apoptotic pathway and might serve as a potential solution to prevent traumatic peritendinous adhesion.