A Water‐Soluble PVA Macrothiol Enables Two‐Photon Microfabrication of Cell‐Interactive Hydrogel Structures at 400 mm s ⁻¹

Two-photon polymerization (2PP) has garnered increasing attention for engineering hydrogels with tailored architectures and controlled cellular responses. However, current 2PP strategies typically rely on (meth)acrylated proteins and inefficient chain-growth crosslinking mechanisms. Although thiol-ene photo-click reactions can enhance 2PP efficiency, commercial water-soluble thiol crosslinkers (e.g., DTT-dithiothreitol) tend to form intramolecular loops and introduce structural defects due to their short molecular length. As a result, high polymer concentrations (often up to 20%-50%) are required to achieve satisfactory print fidelity. Here, we develop a series of water-soluble, polyvinyl alcohol macromolecular thiol (PVASH, bearing 10-35 thiol groups) for fast high-fidelity hydrogel microfabrication via 2PP. A two-step synthesis yields PVASH with tunable degrees of substitution and excellent water-solubility. Compared to DTT and polyethylene glycol di-thiol, PVASH-based hydrogels exhibit reduced swelling, enhanced mechanical properties, and significantly improved printing fidelity. Notably, several complex hydrogel structures are fabricated at laser power as low as 20 mW and high scanning speeds of up to 400 mm s^-1, achieving sub-micron feature size at 3% polymer concentration. After biofunctionalization with RGD motifs, the micro-scaffolds support cell infiltration, adhesion, proliferation, and osteogenic differentiation. Altogether, this work reports a new strategy for 2PP microfabrication of cell-interactive hydrogel structures with unprecedented printing efficiency and precision.