Brushite (CaHPO4·2H2O) is a calcium phosphate mineral that is a common component of human kidney stones, particularly as a precursor in Randall’s plaque that promotes the crystallization of hydroxyapatite and calcium oxalate (CaOx) in renal calculi. Compared to other kidney stone constituents, brushite stones recur more frequently, making their prevention and treatment particularly important and challenging. As the prevalence of calcium phosphate kidney stones has increased over the past three decades, research has focused on inhibitors that can regulate brushite crystallization, including metal ions, small molecules, and proteins. Citrate (CA), a natural component of urine, has demonstrated inhibitory effects on CaOx crystallization, while hydroxycitrate (HCA), a structural analog with an additional hydroxyl group, has been found to be even more effective. In this presentation, we will discuss our investigations into the impact of both molecular modifiers on brushite crystallization, employing bulk crystallization assays, nucleation studies, in situ microfluidics and atomic force microscopy (AFM). Our results show that both CA and HCA influence crystal morphology and inhibit nucleation and growth, with HCA exhibiting significantly stronger effects. Studies using binary combinations of the two modifiers reveal antagonistic cooperativity at low concentrations, while HCA dominates growth inhibition at higher modifier concentrations. Microfluidic experiments demonstrated that CA weakly inhibits brushite growth in a direction-dependent manner, whereas HCA strongly suppresses growth across all crystallographic directions. AFM imaging further revealed that at high supersaturation, CA and HCA act similarly, but at low supersaturation, HCA uniquely disrupts step growth. Collectively, these findings offer key insights into the mechanisms governing brushite crystallization and suggest potential strategies for mitigating kidney stone formation.
