Inkjet biofabrication of electroactive rGO/growth-factor micropatterns on electrospun PLCL for guided neural cell responses

Biofabrication of neural interfaces requires spatial control over conductive, biochemical, and topographical cues at the cell-contacting surface. Here, we report and validate an in vitro biofabricated screening platform based on electrospun poly(lactide-co-ε-caprolactone) (PLCL), in which graphene oxide (GO)/growth-factor precursor inks are inkjet-printed and reduced with ascorbic acid to form reduced graphene oxide (rGO)/growth-factor micropatterns. The platform was used to screen printing-layer number, nozzle configuration, and electric-field strength by integrating band-width fidelity, continuity, overspray, and apparent conductivity with neural-cell readouts. A 4-layer double-nozzle condition produced a core printed-band width of 204.8 µm, close to the designed 200 µm width, with 94.5% continuity and 0.50% overspray, while providing an apparent conductivity of 0.432 ± 0.038 S/cm, approximately 78.8% of the 10-layer condition. The same workflow localized nerve growth factor (NGF) or neuregulin-1 at the basal cell-material interface. Fluorescence-assisted imaging verified post-reduction pattern retention, while ELISA quantified immunoreactive recovery and cumulative release. Across the tested models, the printed interface and stimulation conditions were associated with changes in neural cell behavior. In PC-12 cultures, 150 mV/cm direct-current stimulation was associated with greater βIII-tubulin-positive neurite-like outgrowth and alignment than W/O ES or 300 mV/cm, reaching 19,060 ± 2,417 µm total neurite length per field at day 21. Dorsal root ganglion neurons extended aligned TUJ1-positive neurites along the printed tracks, and S16 Schwann cells expanded on neuregulin-1/rGO-patterned substrates under the same stimulation condition. RT-qPCR and western blotting showed higher neuronal regeneration-associated and Schwann-cell-associated marker levels under 150 mV/cm than under unstimulated or higher-field conditions. These results establish an in vitro screening platform for evaluating printing parameters and defined topographical, electrical, and biochemical cues within the tested neural cell models.