3D Printing of Proteins Via Temperature‐Dependent Amyloid Aggregation

Abstract 3D printing of protein materials for creating bioactive scaffolds has attracted significant interest. However, achieving controllable and stable printing while replicating the ordered structure found in natural protein materials remains a key challenge. Herein, a universally applicable temperature‐dependent protein aggregation (TPA) strategy is reported to manipulate the unfolding, relaxation, and reorganization of protein chains to enable the 3D printing of amyloid‐like proteins. The disruption of internal disulfide bonds induces the unfolding and relaxation of protein, leading to the formation of an amorphous protein sol through chain entanglement as primary cross‐linking points. These relaxed protein chains further aggregate through conformational transition to initiate amyloid‐like protein aggregation rich in β‐sheet structures at high temperature, resulting in a protein gel with β‐sheet nanocrystals serving as secondary cross‐linking points. This gel facilitates the stable and precise extrusion‐based 3D printing of proteinaceous scaffolds with a hierarchically ordered structure. The biomedical potential of this 3D‐printed protein scaffold is preliminarily validated through its biomineralization capability and following application in bone tissue regeneration using rat skull defect models. This strategy demonstrates a facile approach for the controllable structural aggregation of proteins in vitro and holds great potential in the field of protein‐based scaffolds.