Hybrid electro-assisted bioprinting of three-layer nerve guidance conduits with aligned cell-laden micropatterns for peripheral nerve regeneration

Conventional cell-laden electro-direct writing (CEDW) technology faces challenges in terms of balancing printability, mechanical integrity, and biocompatibility. As a result, its application in neural tissue engineering remains limited. To overcome this challenge, this study develops a temperature-regulated cell-laden electro-direct writing (TCEDW) technology that achieves stage-specific rheological modulation through precise cylinder temperature control, thereby decoupling printability from biocompatibility and enabling high-resolution (36.05 µm) cell printing. Using Schwann cell-laden alginate/gelatin methacryloyl/polyethylene oxide (alginate/GelMA/PEO) bioinks, TCEDW constructs were fabricated with both improved structural fidelity and preserved cell viability. In vitro co-culture of Schwann cells and PC12 cells demonstrated that TCEDW microfilaments significantly promoted neurite alignment and maturation by providing synergistic topographical and biochemical cues. Gene expression analysis revealed that TCEDW scaffolds activated axon guidance and cGMP-PKG signaling pathways while suppressing proliferation programs to promote PC12 neuronal differentiation. Furthermore, a hybrid electro-assisted bioprinting approach integrating melt electro-writing, electro-spinning, and TCEDW was employed to fabricate three-layer composite nerve guidance conduits (NGCs). In vivo investigations demonstrated that the composite NGC platform promoted nerve fiber regrowth, Schwann cell migration, myelination, and functional recovery. This study introduces a temperature-controlled TCEDW platform for precise cell-laden microarchitecture fabrication, offering a new strategy for enhancing peripheral nerve regeneration.