3D Bioprinting of Bio-orthogonally Cross-Linked Microporous Scaffolds Using Monodisperse Hydrogel Microparticles...

Three-dimensional (3D) bioprinting presents unparalleled capabilities for probing long-term mechanotransduction mechanisms at the tissue level, yet existing technical constraints in developing efficient, cytocompatible cross-linking methods and the mismatch between bioink properties and native tissue physicochemical characteristics hinder the realization of its transformative potential. We introduce a strategy featuring encapsulated, flexible, and physiologically relevant 3D cell-laden microparticle-annealed scaffolds that recapitulate the mechanical microenvironment of native tissues. Our approach is a rapid bio-orthogonal cross-linking system using trans-cyclooctene (TCO)-tetrazine (Tz) click chemistry, which achieves highly efficient and low-toxicity biocompatible polymerization while preserving scaffold integrity. High-throughput production of monodisperse hydrogel microparticles (HMPs) is enabled by a microchannel array (MiCA) device, allowing precise bioink rheological tuning to optimize printing fidelity and spatial resolution. The resulting centimeter-scale RAW 264.7-laden constructs (>10⁵ cells) demonstrate sustained proliferative activity over 10 days and expected response to LPS stimulation. Integrated transcriptomic and proteomic analyses of HEK293T-laden constructs identified mechanoresponsive genes and proteins that show emerging migratory patterns and cell-matrix interactions indicative of mechanotransduction-driven adaptation. This versatile platform not only facilitates the systematic investigation of collective cellular mechanoresponses but also opens new avenues for developing mechanically adaptive tissue models.