Anionic collagen processed by alkaline hydrolysis: rheological, biological, and 3D printability assessment

Abstract

Collagen is the most abundant structural protein in the extracellular matrix, and its biocompatibility can be enhanced by chemical modification into a negatively charged matrix. This study investigates the effect of alkaline hydrolysis time (24, 48, 72, and 96` h) on the rheological behavior and processability of anionic concentrated collagen solutions extracted from bovine tendon. By systematically modulating hydrolysis time, we demonstrate how increasing negative charge density drives a rearrangement of the collagen network, resulting in more elastic and shear-thinning behavior, as evidenced by higher storage moduli, increased zero-shear viscosity, and enhanced creep-recovery response. Among the tested formulations, the 72-h hydrolyzed collagen exhibited the most balanced rheological profile and enabled extrusion-based 3D printing with high fidelity, achieving geometry preservation of 104 ± 4% (star angle), 98 ± 2% (scaffold area), and effective macroporosity of 84%. Longer hydrolysis led to decreased scaffold pore size and increased absorption of phosphate-buffered saline. All materials were non-cytotoxic and supported cell adhesion (58.74—74.89%). Overall, this work highlights how rheological characterization can guide the design and processability of collagen-based bioinks, linking structural and mechanical properties to 3D printing performance and biological outcomes.