Hydroxylation Mechanism of Hydroxyapatite Nucleation with Aspartate Assistance under Confinement Revealed by Machine Learning Molecular Dynamics

Hydroxyapatite (HAp) nucleation requires incorporation of hydroxyl (OH), yet how hydroxylation emerges, and OH groups become stabilized during Ca-P assembly, particularly under nanoconfinement and with organic molecules, remains unclear. Here, we employ machine learning interatomic potentials (MLIPs) in molecular dynamics (MD) simulations to track hydroxylation pathways and cluster evolution in bulk solution and between graphene sheets containing l-aspartate (Asp). Hydroxylation occurs mainly through proton transfer (PT) events that directly generate Ca-OH, ∼10³ times more frequently than capture of existing free OH^- from solution, consistent with Grotthuss-type PT. Graphene confinement suppresses the emergence of HAp-relevant, three-Ca-coordinated hydroxyl motifs. Under confinement, l-Asp reorganizes local ion distributions and stabilizes Ca-rich microenvironments, producing the most persistent planar three-Ca-coordinated OH motifs and more highly coordinated Ca, thereby forming more HAp-like early nuclei. Our results support a nucleation sequence in which a CaP framework forms first, hydroxylation proceeds continuously, and under confinement, acidic amino acids later select and stabilize HAp-like hydroxyl coordination, clarifying HAp formation in synthetic and biological nanoenvironments.