Culture Growth Phase-Dependent Influence of Extracellular Vesicles Derived from Stem Cells from Human Exfoliated Deciduous Teeth on Oral Mucosa Cells Proliferation in Paracrine Co-Culture with Urethral Epithelium: Implication for Urethral Reconstruct

Urethral stricture is a disease of fibrotic narrowing that compromises the urethral mucosa and spongiosum. Oral mucosal graft urethroplasty delivers excellent outcomes in complex cases, yet its procedural demands restrict availability beyond specialized centers. Endoscopic transplantation of oral mucosa has been proposed; while feasibility is shown, clinical efficacy remains suboptimal. We asked whether extracellular vesicles from stem cells of human exfoliated deciduous teeth (SHED-EVs) promote oral mucosa fibroblast (OMF) growth under urethra-mimetic paracrine conditions and whether culture growth phase tunes EV function. SHED-EVs were collected during logarithmic (SHED-EV-L) or stationary (SHED-EV-S) phases under xeno-free conditions, isolated by a standardized workflow, and characterized by nanoparticle tracking analysis. miRNA cargo was profiled with a human miRNA microarray platform and normalized for comparative analyses. OMF proliferation was quantified in a horizontal indirect co-culture with urethral epithelial cells using incubator-based time-lapse imaging. SHED-EV-L produced a sustained pro-proliferative effect across 24-96 h, whereas SHED-EV-S showed a weaker early effect with a late catch-up; both exceeded vehicle at 96 h. Fibrosis-related miRNA heat maps showed culture growth phase-dependent patterns: SHED-EV-L displayed relatively higher signals for miR-31-3p, miR-146b-3p, several let-7 members, and selected miR-181 isoforms, whereas SHED-EV-S showed a marked relative increase of miR-486-3p; miR-21, miR-99/100, and miR-205 were broadly comparable between phases. These findings indicate that culture growth phase is a practical design lever that orients SHED-EV cargo and function, supporting phase-matched formulations for adjunctive transurethral applications and motivating in vivo validation and manufacturing-oriented quality controls.