Two-Photon Lithography-based fabrication of 3D fibered scaffolds: addressing anisotropic voxel constraints for the design of bio-inspired scaffolds

Two-photon polymerization is a high-resolution additive manufacturing technique capable of fabricating complex 3D microstructures for cell culture applications. However, voxel-induced anisotropy and proximity effects often result in geometric distortions, limiting the ability to create isotropic scaffolds that accurately mimic the extracellular matrix. In this study, we implemented design strategies to fabricate isometric 3D scaffolds with controlled beam thicknesses of 3, 5, 7, and 10 µm, and correlated these geometric variations with the behavior of NIH 3T3 fibroblasts. We systematically characterized voxel anisotropy and proximity effects, demonstrating that careful optimization of laser power, scanning speed, and line spacing is essential for achieving isometric beam geometries. Our results reveal that beam thickness progressively modulates nuclear volume in 3D-cultured fibroblasts, with thicker beams promoting nuclear expansion consistent with increased adhesion area, reaching a plateau between 7 and 10 µm. Cells on flat glass showed markedly larger nuclear volumes than all 3D conditions, confirming the role of 3D confinement in reducing cell spreading. This work provides a practical methodology for fabricating and characterizing isometric TPP scaffolds, identifying key fabrication parameters, including voxel anisotropy, proximity effects, and depth-dependent aberrations, that must be controlled to achieve structural fidelity, and demonstrates that geometric precision is a critical determinant of cell behavior in 3D culture models.