Dityrosine photocrosslinking of native collagen bioinks for controlled shape-fidelity of bioprinted cardiac tissue constructs: probing the interplay between fibrillogenesis and covalent bond formation

Abstract Collagen bioinks are widely used in biofabrication, but their relatively soft mechanical properties can lead to structural instabilities under cell-generated contraction forces. While synthetic functional groups can be conjugated for covalent crosslinking, these methods often disrupt natural protein fibrillogenesis, thereby compromising collagen fibre architecture. This work presents a strategy for the direct covalent stabilisation of native collagen bioinks with dityrosine bonds via visible-light photocrosslinking with ruthenium (Ru) and sodium persulfate (SPS), avoiding the need for polymer pre-functionalisation. Multimodal characterisation, including high-resolution microscopy, spectroscopy, mass spectrometry, and nanoindentation, identified photocrosslinking conditions that enhance collagen fibrillogenesis and reduce off-target polymer oxidation. Interestingly, the biofabrication process itself affected ultimate collagen fibre architecture, with shear-induced alignment during extrusion enhancing fibril proximity and self-assembly, overcoming inhibitory effects the crosslinkers had on fibrillogenesis via ionic and electrostatic interactions. Leveraging these insights, embedded bioprinting was used to fabricate cardiac constructs with high cell viability (>80%), where dityrosine crosslinking could be tuned to modulate geometric shape changes under cell-generated forces (1-15% shrinkage). Finally, the platform was used to bioprint anatomically accurate double-ventricle human heart models with robust shape fidelity. This research establishes a versatile photocrosslinking framework for bioprinting cardiac constructs with tunable shape stability using native collagen bioinks.