USC-Derived Small Extracellular Vesicles-Functionalized Scaffolds Promote Scarless Vaginal Defect Repair via Delivery of Decorin and DUSP3 Proteins.

Background: Scar formation following large-area vaginal defects post-vaginoplasty is a major clinical challenge. Compared to skin scars, vaginal scars can lead to pain during intercourse and urinary difficulties, severely impacting quality of life. Small extracellular vesicles (sEVs) encapsulate diverse bioactive components, making them potential therapeutic agents. Designing functional scaffolds that incorporate sEVs is a promising approach for scarless vaginal defect repair. Methods: sEVs-loaded scaffolds were developed through electrostatic interactions between negatively charged sEVs secreted by urine-derived stem cells (USC-sEVs) and positively charged human acellular amniotic membranes. The efficacy of sEVs-loaded scaffolds in the treatment of vaginal defects in rabbits was assessed by histological analysis. Immunofluorescence staining, Western blot, qRT-PCR and collagen gel contraction analyses were conducted to evaluate the antifibrotic effects of USC-sEVs. RNA sequencing was employed to elucidate the underlying mechanisms involved. LC‒MS/MS analysis was used to identify candidate upstream proteins in USC-sEVs. Results: In vivo experiments demonstrated that the sEVs-loaded scaffolds promoted scarless healing of vaginal defects in rabbits by modulating collagen deposition, reducing fibrosis, and diminishing inflammation. In vitro experiments revealed that USC-sEVs significantly inhibited the proliferation, collagen production, and activation of fibroblasts with a fibrotic phenotype, indicating the antifibrotic properties of USC-sEVs. Transcriptome and Western blot analyses revealed that USC-sEVs treatment inhibited fibrosis by downregulating the TGF-β and p38 MAPK signaling pathways. LC‒MS/MS analysis identified 2653 proteins encapsulated in USC-sEVs. Western blot analysis revealed that decorin, an inhibitor of the TGF-β signaling pathway, and DUSP3, a negative regulator of p38 phosphorylation, were enriched in USC-sEVs and could be transferred to fibroblasts. Conclusion: USC-sEVs inhibited fibrosis and promoted scarless healing by delivering decorin and DUSP3 proteins, which regulate the TGF-β and p38 MAPK signaling pathways, respectively. This study highlights the potential of sEVs-loaded scaffolds as a promising strategy for scarless vaginal repair following vaginoplasty, offering a novel approach for regenerative medicine with significant translational potential for clinical application.