Two-photon lithography-based fabrication of 3D fibrous scaffolds: Addressing anisotropic voxel constraints for the design of bio-inspired scaffolds

Two-photon polymerization enables the fabrication of complex 3D microstructures at sub-micrometer resolution, making it a powerful platform for cell culture scaffold fabrication. However, voxel-induced anisotropy and proximity effects often result in geometric distortions that introduce unintended structural directionality, limiting the ability to decouple scaffold geometry from fabrication artifacts in cell behavior studies. In this work, we characterized these phenomena and developed practical design strategies, including elliptical cylindrical shell geometries with uniform line spacing, optimized writing path strategies, and depth-dependent power compensation, to fabricate isometric 3D cage scaffolds with controlled beam width. SEM characterization confirmed aspect ratios close to unity across all beam sizes and orientations, indicating that the adopted strategies were effective in achieving the targeted isometric geometries. NIH 3 T3 fibroblasts cultured within these scaffolds exhibited a progressive increase in nuclear volume with beam width, plateauing between 7 and 10 μm, consistent with differences in available adhesive contact area. This work provides a practical methodology for fabricating isometric scaffolds using two-photon polymerization and establishes beam width as a controllable geometric parameter for investigating geometry-dependent cell responses in 3D culture models.