Sustained delivery of 3D-bioprinted stem cell-derived small extracellular vesicles promotes neural regeneration following traumatic brain injury via the CREB1/PGC-1α/FNDC5 axis

Background Clinical translation of stem cell-derived small extracellular vesicles (sEVs) for traumatic brain injury (TBI) is restricted by low yield in 2D culture and rapid in vivo clearance. Objective To develop an in situ sustained delivery system (3D-sEVs@Gel) encapsulating 3D-bioprinted adipose stem cell (ADSC)-derived sEVs (3D-ADSC-sEVs) within GelMA (Gelatin methacryloyl) hydrogel for long-term functional repair after TBI. Methods SEVs derived from coaxially bioprinted 3D-ADSCs were isolated, and a GelMA hydrogel-based delivery system was subsequently constructed. Following orthotopic transplantation of the 3D-sEVs@Gel system into the lesion site after TBI, functional recovery, neural stem cell (NSC) proliferation and lineage-associated responses, and the underlying molecular mechanisms were systematically evaluated through behavioral tests, immunofluorescence, multi-omics, and pharmacological inhibition. Results SEVs generated using the coaxial alginate-microfiber culture system exhibited an approximately eightfold higher yields and protein loading compared with those obtained from 2D culture. In vivo , 3D-sEVs@Gel prolonged local retention, reduced tissue loss, and significantly improved long-term sensorimotor coordination and spatial memory following TBI. In addition, treatment increased the numbers of proliferating cells co-expressing neural progenitor-, neuronal-, and oligodendroglial-lineage-associated markers within the examined neurogenic regions. Mechanistically, 3D-sEV treatment increased nuclear CREB1 levels in NSCs, promoting PGC-1α transcription and subsequent FNDC5 expression, thereby supporting endogenous neurogenesis and functional neural repair. Conclusion The 3D-sEVs@Gel system promotes chronic neurological reconstruction post-TBI by modulating NSC proliferation and neural lineage-associated responses through the CREB1/PGC-1α/FNDC5 pathway, offering a promising cell-free strategy for neural repair and tissue engineering.