Heat-Suppressing Projection Two-Photon Lithography Enables High-Throughput Sub-Micrometer Manufacturing of Biopolymer Hydrogels for Tissue Engineering.

Native tissues exhibit complex, multiscale hierarchies ranging from centimeter-scale organization to sub-micrometer extracellular matrix (ECM) topographies. While two-photon polymerization (TPP) lithography provides the sub-micrometer precision essential for mimicking the ECM, traditional point-scanning TPP is constrained by prohibitively low fabrication efficiency. Although projection two-photon lithography (P-TPP) significantly enhances throughput, its application in biopolymer hydrogels is severely hindered by intensive localized heat accumulation, often resulting in material charring and compromised bioactivity. In this study, we developed a heat-suppressing P-TPP platform that overcomes these thermal limitations. By employing a low-exothermic Type II photoinitiating system, we effectively mitigate thermal damage during high-power polymerization, enabling the high-fidelity fabrication of hydrogel micro-units with sub-micrometer resolution. This technical advancement improves production throughput by 3-4 orders of magnitude compared to conventional point-scanning TPP. To bridge the gap between micro-precision and macro-scale tissue engineering, we further integrated this platform with extrusion-based printing, a process termed integrated lithography and extrusion additive production. This multiscale manufacturing approach allows for the assembly of engineered micro-units into sophisticated macroscopic hydrogel constructs that provide critical structural cues for cell alignment and functional tissue maturation. Our platform offers a scalable paradigm for the fabrication of multiscale hydrogels tailored for advanced biomedical applications.