Laser-engineered microtopographies for probabilistic control of cell shape and adhesion: from predictability to exploration

Fibroblast shape directs cell function and tissue organization, yet systematically controlling cell morphology requires highly reproducible surface topographies, which have so far been lacking. Here, we demonstrate a strategy to precisely guide fibroblast cell shapes and adhesion through design of simple and reproducible microtopographies fabricated by laser direct writing via two-photon polymerization of IP-Dip photopolymer. The engineered microtopographies consisted of parallel lines (2–4 μm spacing and 1028 ± 68 nm in height) and sets of intersecting lines spaced 2 μm apart and overlapped at 11.5°, 22.6 and 33.4°, respectively. Single-cell analysis of murine L929 fibroblasts revealed clear geometry-dependent phenotypes: flat substrates promoted polygonal shapes (circularity 0.65 ± 0.07; solidity 0.89 ± 0.04; vinculin 511,867 ± 68,060 a.u.), parallel lines induced elongated spindle-like morphologies (circularity 0.49 ± 0.05; solidity 0.93 ± 0.04; vinculin 469,604 ± 111,409 a.u.), and intersecting lines generated stellate cells (circularity 0.48 ± 0.07; solidity 0.77 ± 0.05; vinculin 336,256 ± 94,193 a.u., p < 0.01 vs parallel). The microtopographies also induced rounded transient morphologies in the seeded fibroblasts, with a higher number of rounded cells with filopodia-like protrusions (15.4 and 14 cells/100 μm 2 on parallel and overlapped lines, respectively) relative to flat surfaces (6.2 cells/100 μm 2 ), whereas rounded cells with bleb-like protrusions predominated on flat substrates (7.8 cells/100 μm 2 ) as compared to all other topographies investigated. Unlike conventional approaches focused on predictable geometry-driven cell behavior, our study proposes a semi-predictive model in which microtopographies influence cellular adaptation without fully determining it.