Extracellular vesicles derived from salivary gland stem cells cultured on microwell scaffolds loaded with WNT3A promote the recovery of salivary gland function damaged by radiation via the YWHAZ-PI3K-AKT pathway.

Salivary gland (SG) stem cell-derived extracellular vesicles (EVs) are promising agents for regenerative therapy, but efficient production and targeted delivery remain key challenges. We developed a WNT3A-releasing double-layered microwell scaffold by integrating WNT3A-loaded poly(D,L-lactide-co-glycolide) (PLGA) nanofibers with a polycaprolactone (PCL)-based microwell array. This 3D platform promotes salivary gland epithelial stem cell (sgEpSC) spheroid formation and sustained biochemical stimulation. EVs derived from four culture conditions (2D dish, 3D Microwell, 3D PLGA-Microwell, and 3D WNT-Microwell) were analyzed for yield, purity, and therapeutic efficacy. The WNT-Microwell system enabled stable spheroid formation and sustained WNT3A release over 7 days. sgEpSCs cultured on this platform produced significantly higher EV yields than other conditions. In a murine model of radiation-induced SG damage, retroductal injection of EVs from 3D spheroids cultured in WNT3A-releasing microwells (3D(WNT)-EVs) reduced apoptosis, preserved acinar structures, and restored saliva secretion more effectively than other groups. In irradiated human SG organoids, 3D(WNT)-EVs increased organoid size, mucin production, and suppressed cleaved caspase-3. Proteomic analysis identified YWHAZ (14-3-3ζ/δ) as a key regenerative cargo. Functional assays showed that EV-mediated delivery of YWHAZ activated PI3K-AKT signaling, enhanced SG progenitor proliferation, and mitigated radiation-induced damage. WNT-Microwell scaffolds enhance the yield and regenerative efficacy of SG-derived EVs. YWHAZ-enriched EVs promote SG repair via PI3K-AKT activation, offering a promising strategy for scalable, cell-free regenerative therapy in SG dysfunction.