Diffusion-Driven Promotion of Calcium Oxalate Monohydrate Crystallization on Brushite: Wide-Field In Vitro Insights into Protein-Dependent Stone-Forming Environments

Abstract

Despite substantial advances in understanding urinary stone formation, including mechanisms of calcium phosphate (CaP) crystallization, tissue infiltration, and stone development via Randall’s plaques, the global prevalence of urolithiasis continues to rise and current knowledge has only limited impact on clinical outcomes. This study focuses on the CaP phase, particularly brushite, and re-examines its role under physiologically relevant conditions as a potential key to more effective prevention. The aim was to elucidate how reactions proceed near the surfaces of brushite crystals that can present in urine, and how these reactions are modulated by coexisting proteins, using wide-field optical observation combined with quantitative modeling. Single-crystal brushite was immersed in a model solution pre-equilibrated with calcium oxalate monohydrate (COM), and the time evolution of the Ca²⁺ concentration field associated with brushite dissolution was evaluated by in situ microscopy and one-dimensional diffusion analysis. The results demonstrated that brushite dissolution generates a critical Ca²⁺ supersaturation zone extending several hundred micrometers from the crystal surface, within which COM nucleation occurs. Upon addition of bovine serum albumin (BSA) as a model protein, the average growth of COM was inhibited, whereas the local frequency of COM nucleation increased markedly in regions of the brushite surface with high step and kink densities, yielding dense, rigid COM−brushite composites. These findings indicate that brushite crystals function not merely as apatite precursors, but as localized “reaction fields” that spatially control calcium oxalate nucleation, aggregation, and consolidation. Furthermore, urinary proteins can, through these fields, act not only as inhibitors but also as local promoters of stone formation.