| OBJECTIVESTendon injury healing is one of the knotty problems to be solved in hand surgery.Adhesion seems to be unavoidable during tendon healing.Exploring therapeutic strategies to promote tendon healing and prevent tendon adhesions urgently requires innovative theoretical guidance and technical support.In recent years,exosomes hold great potential as a new generation of cell-free therapeutic agents in the field of tissue repair.With its unique advantages,HUMSC has gradually become one of the most appealing exosomal donors.This project intended to explore whether HUMSC-Exos could promote tendon healing and prevent tendon adhesion through in vivo and in vitro experiments,and explore potential mechanisms of action to provide new treatment strategies for tendon injury healing.METHODSIn the first part of the study,a rat Achilles tendon defect healing model was constructed in vivo,and the localized application of HUMSC-Exos was used to evaluate tendon healing.TDSCs were used as cell model in vitro to evaluate the effects of HUMSC-Exos on cell proliferation and gene expression.Mi RNA sequencing and q-PCR analysis confirmed differential miRNAs.Specific miRNA agonist(agomir-29a-3p)transformed HUMSC-Exos and obtained modified exosomes to verify its efficacy and related mechanisms of action.In the second part,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 TGF-?1 and/or HUMSC-Exos in vitro,and cell proliferation,apoptosis and gene expression were measured.Mi RNA sequencing and q-PCR 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.In the third part,the rat Achilles tendon injury adhesion model was constructed in vivo,and the tendon adhesion was evaluated by topical application of the antitumor drug HCPT.After treatment with TGF-?1 and/or HCPT in vitro,rat fibroblast cell lines were tested for proliferation,apoptosis and gene expression.Salubrinal and si RNA/sh RNA were employed to verify whether the effect of HCPT depends on the ERS signaling pathway.In the fourth part,HUMSC was pre-processed with HCPT to obtain modified HUMSC-Exos(HCPT-Exos).The effects of HCPT-Exos and HUMSC-Exos on the proliferation,apoptosis and gene expression of rat fibroblasts were evaluated in vitro.Salubrinal was used to verify whether the superior effect of HCPT-Exos depends on the ERS signaling pathway.Then,we observed that topical application of HCPT inhibited tendon adhesions in rats.HCPT induced apoptosis of rat fibroblasts in vitro and inhibited TGF-?1-induced cell proliferation and the expression of COL3A1 and ?-SMA.After pretreatment of fibroblasts with ERS inhibitor salubrinal and ERS pathway(PERK,IRE-1 and ATF-6)RNA interference technology,the anti-fibrosis effect of HCPT was inhibited,indicating that its anti-fibrosis effect might depend on ER-related apoptosis.RESULTSLocal application of HUMSC-Exos promoted healing of the Achilles tendon in rats,and promoted TDSCs cell proliferation and expression of tendon markers.Mi RNA sequencing showed that miR-29a-3p was enriched in HUMSC-Exos.Specific miR-29a-3p agonists transformed HUMSC to obtain modified HUMSC-Exos with high expression of miR-29a-3p.Further in vivo and in vitro experiments confirmed its enhanced tendon-promoting healing effect,which may be related to activating TGF-?1/Smad3/m TOR signaling cascade.Secondly,the Achilles tendon adhesions in rats were alleviated after the local injection of HUMSC-Exos.HUMSC-Exos suppressed TGF-?1-induced cell proliferation and expression of COL3A1 and ?-SMA in rat fibroblasts.Mi RNA sequencing and q-PCR confirmed low expression of miR-21a-5p in HUMSC-Exos.Specific miR-29a-3p antagonists modified HUMSC to obtain HUMSC-Exos with low expression of miR-21a-5p,and subsequent in vitro experiments confirmed its enhancement anti-adhesion/fibrosis effects,which may be related to the manipulation of p65 activity.Finally,HCPT pretreated HUMSC and harvested modified HCPT-Exos,which were superior to HUMSC-Exos in inhibiting cell proliferation,promoting apoptosis and inhibiting fibrosis gene expression.Salubrinal pretreatment partially eliminated the advantages of HCPT-Exos,indicating that the superior antifibrotic effect of HCPT-Exos might depend on the ERS signaling pathway.CONCLUSIONSHUMSC-Exos promoted tendon healing and prevented tendon adhesions,and its mechanism of action may be closely related to differentially expressed miRNAs in exosomes.Engineering or drug-modified exosomes could further enhance its role,which provided theoretical basis and new treatment strategies for tendon injury repair,and provided strong support for clinical-grade tendon injury treatment. |
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