Synergistic effects of graphene microgrooves and electrical stimulation on M2 macrophage polarization.

Macrophages play a crucial role in host response and wound healing, with M2 polarization contributing to the reduction of foreign-body reactions induced by the implantation of biomaterials and promoting tissue regeneration. Electrical stimulation (ES) and micropatterned substrates have a significant impact on the macrophage polarization. However, there is currently a lack of well-established cell culture platforms for studying the synergistic effects of these two factors. In this study, we prepared a graphene free-standing substrate with 20 μm microgrooves using capillary forces induced by water evaporation. Subsequently, we established an ES cell culture platform for macrophage cultivation by integrating a self-designed multi-well chamber cell culture device. We observed that graphene microgrooves, in combination with ES, significantly reduce cell spreading area and circularity. Results from immunofluorescence, ELISA, and flow cytometry demonstrate that the synergistic effect of graphene microgrooves and ES effectively promotes macrophage M2 phenotypic polarization. Finally, RNA sequencing results reveal that the synergistic effects of ES and graphene microgrooves inhibit the macrophage actin polymerization and the downstream PI3K signaling pathway, thereby influencing the phenotypic transition. Our results demonstrate the potential of graphene-based microgrooves and ES to synergistically modulate macrophage polarization, offering promising applications in regenerative medicine.