Localized enzyme-regulated calcium phosphate mineralization of individual collagen fibrils: Exploring intra- and extra-fibrillar pathways.

The biogenic calcium phosphate mineralization is a highly regulated and spatially confined process occurring within the extracellular matrix (ECM), in which matrix vesicles play a central role through localized enzymatic regulation of ion homeostasis. Reproducing this level of spatial and temporal control in vitro remains challenging, as conventional strategies rely on bulk supersaturation and fail to recreate the localized ion homeostasis and compartmentalization that govern physiological mineral formation. Herein, we introduce a biomimetic system that associates collagen fibrils with localized enzymatic activity to drive mineralization in a controlled ECM-like environment. By integrating compartmentalized ion generation with dynamic homeostatic control, the system enables calcium phosphate formation in the immediate vicinity of individual collagen fibrils. Using real-time atomic force microscopy, we directly visualize mineral formation, growth, and nanomechanical evolution at the single-fibril level. Strikingly, we demonstrate mineralization within both intrafibrillar and extrafibrillar compartments, revealing spatial control particularly challenging to achieve in biomimetic systems. These findings highlight the relevance of the proposed biomimetic system for controlling collagen mineralization in space and time and for exploring both intra- and extrafibrillar mineralization pathways. The robustness of this versatile system provides mechanistic insights into enzymatic ion regulation and fibrillar confinement,. It offers a powerful strategy to explore physiological biomineralization pathways and to design advanced bioinspired mineralized materials.