4D printing of stretchable nanocookie@conduit material hosting biocues and magnetoelectric stimulation for neurite sprouting

The study addresses the challenge of delivering on-demand biochemical and electrical cues to program neuron-related cells for peripheral nerve regeneration. While 3D/4D printing enables complex, personalized biomedical structures, few techniques provide controllable surface exposure of conductive nanomaterials and remote, on-demand electrical stimulation to promote neurite sprouting and Schwann cell migration. The research hypothesis is that a 4D-printed, stretchable conduit embedding surface-exposed, conductive nanocookies (NCs) can convert a high-frequency magnetic field into localized electric currents to trigger growth factor release and directly stimulate cells, thereby enhancing neurite outgrowth and nerve regeneration. The work integrates nanoconductors with light-curing 3D printing to expose NCs on scaffold surfaces, providing both topographic cues and magnetoelectric stimulation to address limitations of traditional hydrogels and invasive electrodes in deep nerve stimulation.

Prior work in 4D printing has shown shape/property transformations under stimuli for applications in microrobotics, drug delivery, and tissue engineering. 3D printed dosage forms and scaffolds have demonstrated programmable drug release and growth factor delivery to guide differentiation and vascularization. Electrical stimulation is known to influence cell fate and programming, yet deep nerve stimulation often requires invasive electrodes. Nanoconductors like gold and graphene can be electromagnetized under high-frequency magnetic fields to induce eddy currents for remote control of drug release and to promote osteogenesis and neurogenesis; however, conventional hydrogels embed particles, limiting direct cell contact. Light-curing 3D printing can expose nanoconductors on surfaces for direct cell interaction. Graphene-based elastomers exhibit biocompatibility, conductivity, and neurogenic support. PU-based scaffolds (e.g., Opsite) demonstrate oxygen permeability and mechanical support beneficial for nerve repair. This body of work motivates integrating conductive graphene-derived nanostructures with 3D/4D printed elastomers to provide combined topographical, biochemical, and electrical cues for nerve regeneration.

Materials synthesis: Graphene oxide (GO) was synthesized via a modified Hummers method using graphite, concentrated H2SO4, NaNO3, KMnO4, and H2O2, followed by washing and drying. Nanocookies (NCs), consisting of mesoporous silica/carbon on GO sheets, were prepared by templating with CTAB and organics (octane, styrene), L-lysine, TEOS, and AIBA, followed by washing, drying, calcination at 450 °C under N2, and purification.

3D printing of conduits: A DLP 3D printer (MiiCraft plus, 405 nm LED, 12.5 mW) was used. Base resin: 4-hydroxybutyl acrylate (4-HBA), PU-EO-PO monomer, and photoinitiator (Irgacure 819). NC-loaded resins contained 0.1% or 1% NCs by mass. Resins were degassed by ultrasonication. CAD models were sliced (100 µm layers, 10 s exposure per layer). Printed constructs were postcured under 18 W UVA, washed with deionized water, and sterilized via autoclave (121 °C, 15 psi). Conduits incorporated internal microchannels formed additively.

Characterization: NC structure assessed by TEM; Raman spectroscopy for graphite/GO/NC bonding; DLS for particle size distribution; N2 adsorption-desorption (BET surface area, BJH pore size); SQUID magnetometry for field-dependent magnetization (−40,000 to 40,000 Oe at 6 K and 300 K) and temperature dependence (5–300 K at 50 Oe); XPS for surface composition. Conduit morphology by FE-SEM; mechanical testing per ASTM-D638-14 using dog-bone specimens with 1 kN load cell at 10 mm/min to determine Young’s modulus and ultimate tensile strength.

Cell culture and biocompatibility: HIG-82, N2a, Schwann cells (SCs), and PC12 cells were cultured in standard media with supplements. Cytotoxicity followed ISO 10993-5/12: printed samples (1×1×0.1 cm³) were extracted into serum-containing media; MTT assay measured viability at 570/650 nm. MF exposure for some groups used a 1 MHz, 3.2 kW field for 15 min with the applicator positioned 1 cm above cultures.

Cell imaging: For SEM, cells on matrices were fixed (2.5% glutaraldehyde), postfixed (2% OsO4), dehydrated through graded ethanol, and acetone-treated. For CLSM, cells were fixed (3.7% formaldehyde), permeabilized (0.1% Triton X-100), and stained with DAPI and phalloidin.

NC uptake and MF effects: NCs were labeled with quantum dots (QDs) by co-dissolution in chloroform, mixing (24 h), drying, washing, and centrifugation to remove free QDs. PC12 cells were incubated with NCs/QDs for 0.5–4 h for flow cytometry. For neurite analysis, PC12 cells were exposed to MF (1 MHz, 3.2 kW, 15 min) after 4 h NC incubation and then cultured for 3 days before fixation and staining.

Topographical guidance and differentiation: Conduits with/without microchannels were sterilized and seeded with HIG-82 cells for 1–4 days. Orientation analysis used CLSM images processed in Nikon NIS-elements with Feret angle and Gaussian fitting of circumferential profiles. For PC12 differentiation, control cultures received 100 ng/mL NGF. Experimental groups used sterilized printed planes pre-immersed in 100 ng/mL NGF for 2 days; cells were seeded, then treated with MF (1 MHz, 3.2 kW, 15 min) and cultured 4 days prior to staining.

Animal model and surgery: Male Sprague-Dawley rats (~3 weeks) under IACUC-approved protocols underwent left sciatic nerve transection creating a 10 mm gap near the bifurcation. Repair was with either autograft (inverted, sutured with 9-0, four sutures each side) or 3D-printed conduit. Postoperative care allowed free access to food/water.

Functional and morphological assessments: Walking track analysis recorded at 1 month to compute Sciatic Function Index (SFI) from print length (PL), toe spread (TS), and intermediate toe spread (ITS). Gastrocnemius muscles were excised and weighed bilaterally to compute relative muscle weight. For histology, regenerated nerves were fixed (4% PFA), cryosectioned, and immunostained for βIII-tubulin with FITC secondary and DAPI. Orientation analysis matched that for cell studies. For ultrastructure, samples were processed for TEM (glutaraldehyde, OsO4, ethanol dehydration, Araldite embedding, toluidine blue semithin staining; ultrathin sections poststained with lead citrate/uranyl acetate).

• Magnetoelectric conversion: Under a high-frequency magnetic field (1 MHz, 3.2 kW, 15 min), NCs embedded and exposed on the conduit surface generated eddy currents, providing localized electrical stimulation to cells.
• On-demand growth factor release: NC@conduit with high NGF loading exhibited excellent permeability and MF-triggered, on-demand NGF release.
• In vitro outcomes: MF-activated NC@conduit promoted neuron-related cell responses, including PC12 neurite differentiation and enhanced proliferation; exposed NCs increased surface roughness and improved cell adhesion (SEM/CLSM observations). Microchannels guided cell alignment.
• In vivo nerve regeneration: Following implantation bridging a 10 mm sciatic nerve gap in rats, NC@conduit led to improved axon orientation and increased myelin sheath layers at 1 month postimplantation, indicating enhanced regeneration compared with non-stimulated conditions. Functional assessment employed SFI; muscle mass and histology supported regeneration benefits.
• Mechanical/printing performance: The 4D printed elastomeric conduit was stretchable, with tunable mechanical properties via postcuring, and allowed precise fabrication of internal microchannels exposing NCs on the surface for direct cell interaction.

The findings demonstrate that integrating surface-exposed conductive nanocookies into a 4D-printed elastomeric conduit enables remote magnetoelectric stimulation and controlled growth factor release without invasive electrodes. This addresses key barriers in peripheral nerve repair, namely delivering synchronized topographical, biochemical, and electrical cues to promote neurite sprouting and Schwann cell migration. MF-triggered eddy currents at the scaffold–cell interface, coupled with NGF release, directly enhanced neuronal differentiation in vitro and improved axonal guidance and myelination in vivo after a critical-size sciatic nerve gap. The DLP process is pivotal because it exposes NCs on the surface, maximizing cell contact and stimulus transmission, while built-in microchannels provide directional cues and permeability. Together, these features support a strategy for personalized nerve guides capable of noninvasive, on-demand stimulation to improve regeneration outcomes.

This work presents a 4D-printed, stretchable NC@conduit that combines mesoporous graphene-based nanocookies with a protein-permeable elastomer to deliver topographical guidance, magnetoelectric stimulation, and on-demand NGF release. The platform enhanced neuron differentiation in vitro and improved axonal alignment and myelination in vivo across a 10 mm sciatic nerve gap, offering proof-of-principle for MF-guided nerve regeneration. Future research should quantify dose–response relationships of MF parameters, optimize NC loading and distribution, evaluate long-term functional recovery and biodegradation, and assess safety and efficacy in larger animal models and chronic injury settings.