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

Four-dimensional (4D) printing allows creation of 3D objects with properties that change over time in response to stimuli. This technology holds promise for various applications, including drug delivery, tumor therapy, and tissue engineering. Current challenges in 3D printed tissue regeneration include on-demand biocue release and electrical stimulation to promote neurite sprouting and Schwann cell migration, particularly crucial for peripheral nerve injury (PNI). Electrical stimulation, while effective, often requires invasive electrode implantation. High-frequency magnetic fields (MFs) offer a non-invasive alternative, inducing electric currents (eddy currents) in conductive materials like gold and graphene, which can control drug release and promote neurogenesis. However, direct cell contact with these nanoconductors is challenging with traditional hydrogel methods. This study aims to overcome this by integrating nanoconductors into 3D printed structures to create a biocompatible conduit for peripheral nerve regeneration, using a high-frequency magnetic field for non-invasive stimulation.

Previous research highlights the use of graphene in drug delivery, stem cell differentiation, and tissue engineering due to its biocompatibility and conductivity. 3D printing of graphene elastomers offers excellent electrical and flexible properties, supporting neurogenic differentiation. Studies have also shown the potential of 3D printed polyurethane (PU)-based hydrogels with bioactive ingredients for cartilage regeneration and wound healing. The authors review the existing limitations of current 3D printing techniques in achieving controllable surface roughness and on-demand electric currents in biomicroenvironments to induce various biological processes. The current study builds on these advancements by integrating nanoconductors into a 3D printed structure for enhanced functionality and improved nerve regeneration.

The study involved synthesizing reduced graphene oxide (GO) nanosheets and nanocookies (NCs) using a modified Hummers method and a previously described method, respectively. A digital light processing (DLP) 3D printer was used to fabricate a stretchable conduit (NC@conduit, NC@C) using a bioink composed of NCs and a light-curable polymer. The NCs’ structure and properties were characterized using TEM, Raman spectroscopy, DLS, N2 adsorption-desorption, and SQUID magnetometry. The 3D printed conduit’s mechanical properties were assessed via tensile testing. In vitro biocompatibility was evaluated using MTT assays with various cell lines (HIG-82, Neuro-2a, SCs, PC12). Flow cytometry and confocal laser scanning microscopy (CLSM) were used to analyze cell uptake of quantum dots (QDs)-labeled NCs and cell behavior under MF treatment. In vivo studies involved implanting the NC@C conduit into sciatic nerves of Sprague-Dawley rats after a 10 mm transection. Functional assessment included walking track analysis, gastrocnemius muscle weight measurement, and immunohistochemistry for axon regeneration and myelin formation. Stereographic analysis was performed using electron microscopy.

The fabricated NC@C conduit exhibited excellent stretchability and biocompatibility. In vitro studies demonstrated that MF treatment of NC@C promoted cell proliferation and differentiation. In vivo implantation of the NC@C conduit into transected sciatic nerves of rats showed significant improvement in axon outgrowth and myelin formation at one month post-implantation compared to the autograft group. The functional assessment revealed improved sciatic function index (SFI) values and gastrocnemius muscle weight in the NC@C group. Immunohistochemical analysis confirmed the presence of regenerated axons and myelin sheaths within the conduit. The surface roughness of the exposed NCs on the conduit surface enhanced cell attachment and improved cell guidance.

The findings demonstrate the successful integration of 4D printing, magnetoelectric stimulation, and biocompatible nanocomposites to promote peripheral nerve regeneration. The surface-exposed NCs in the 3D printed conduit facilitate efficient cell adhesion and targeted electromagnetic stimulation, leading to enhanced cell proliferation and differentiation. The on-demand release of growth factors from the NC@conduit further contributes to the improved nerve regeneration outcomes observed in vivo. The study provides a promising approach for the development of novel biomaterials for treating peripheral nerve injuries and other neuronal diseases.

This study successfully demonstrated the efficacy of a novel 4D-printed NC@C conduit for peripheral nerve regeneration. The integration of magnetoelectric stimulation and on-demand growth factor release significantly improved functional recovery in a rat model. Future research could explore different nanocomposite materials, optimization of MF parameters, and clinical translation of this technology.

The study was conducted on a rat model and might not directly translate to human applications. The long-term efficacy of the NC@C conduit requires further investigation. Further studies are also needed to explore the potential effects of the high-frequency magnetic field on surrounding tissues.