Three-Dimensional Gelatin-Based Hydrogel Degradation and Cell Control by Two-Photon Excitation

Light-responsive hydrogel-based cell scaffolds enable noninvasive, high-precision microenvironmental editing through light stimulation. Two-photon excitation using near-infrared lasers has recently gained attention as a less cytotoxic light irradiation method for cells. Two-photon excitation is an optical phenomenon that generates a localized high-energy state through the simultaneous absorption of two long-wavelength photons, enabling precise manipulation at the single-cell level, even within thick gels. In this study, photodegradable gelatin acrylate (Gelatin-PDA) hydrogels incorporating o-nitrobenzylacrylate (PDA) photodegradation units into a gelatin scaffold were degraded via two-photon excitation. Upon light irradiation, the Gelatin-PDA hydrogel was degraded, loosening its network. We demonstrated that it can be precisely decomposed into both two-dimensional and three-dimensional patterns. By light-irradiating the surface and interior of the Gelatin-PDA hydrogel, we controlled the human bone marrow-derived mesenchymal stem cell (hBM-MSC) orientation along the pattern and mouse embryonic fibroblast (NIH3T3) spheroid elongation. In this study, we achieved the dynamic and precise control of the cell elongation direction within a three-dimensional cell scaffold by combining a Gelatin-PDA hydrogel with advanced two-photon excitation technology. This contributes significantly to complex and dynamic biological tissue mimicry and paves the way for advances in regenerative medicine.