3D-bioprinted adipose-derived stem cell-secreted GAS6+-sEVs reprogram microglia polarization and alleviate neuroinflammation in traumatic brain injury

Traumatic brain injury (TBI)-induced neuroinflammation, driven by inflammatory microglial polarization, continues to pose a significant regenerative and clinical challenge. Small extracellular vesicles (sEVs) have demonstrated great potential in mitigating post-TBI inflammation. Nevertheless, the limited yield and efficacy of sEVs produced via conventional two-dimensional (2D) culture systems (2D-sEVs) substantially hinder their clinical applicability. Moreover, effective strategies for the therapeutic application of sEVs in TBI treatment, along with an understanding of their underlying mechanisms, remain largely unexplored. In this study, we employed a 3D coaxial bioprinting method to encapsulate adipose-derived stem cells (ADSCs) within a hydrogel microfiber, facilitating 3D culturing and large-scale production of 3D-sEVs. Additionally, we utilized GelMA hydrogel for the sustained release of 3D-sEVs and evaluated their effects in LPS-activated microglia as well as in a TBI mouse model. Our results demonstrated that 3D culture significantly enhanced sEV production. GelMA improved sEV stability and prolonged sEV release up to 30 days in vivo. Compared to 2D-sEVs, 3D-sEVs offered superior therapeutic benefits. Specifically, 3D-sEVs substantially reduced neuroinflammation and brain tissue loss while accelerating motor function recovery in TBI mice. Furthermore, 3D-sEVs shifted pro-inflammatory microglia toward an anti-inflammatory polarization state, as evidenced by elevated expression levels of IL-4, IL-10, TGF-β, Arg1, and CD206, alongside reduced expression of IL-6, IL-1β, TNF-α, iNOS, and CD86, both in vitro and in vivo. Additionally, 3D-sEVs attenuated chemotaxis and migration in LPS-activated microglia. Further mechanistic exploration through RNA-seq, proteomic profiling, and GAS6 knockdown in 3D-sEVs, revealed that 3D-sEVs deliver growth arrest-specific protein 6 (GAS6) to modulate the transition of microglia from a pro-inflammatory to an anti-inflammatory state, thereby mitigating neuroinflammation following TBI. Our findings underscore the therapeutic promise of sEVs derived from 3D-cultured ADSCs in treating TBI via modulating microglia polarization.