Nucleation of Brushite and Hydroxyapatite from Amorphous Calcium Phosphate Phases Revealed by Dynamic in Situ Raman Spectroscopy

Abstract

Since the 1970s, it has been shown that calcium phosphate crystals nucleate from one or several early amorphous calcium phosphate phases into several in vivo and in vitro systems. However, the precise chemical composition, structure, and transformation mechanism of these amorphous phases remain controversial. Here, we characterize the reaction mechanism and kinetics of formation of two phosphate crystals, brushite and hydroxyapatite, by using in situ Raman spectroscopy in batch reactors at 25 °C. We investigate three pH regimes to control the phosphate speciation in solution and used solutions with or without citric acid, a complexing agent that may stabilize the amorphous phases. As expected, brushite (CaHPO4·2H2O) forms at pH < 9.8. Amorphous calcium phosphate (ACP: CaHPO4·nH2O) with a short lifetime (<2 min) and octocalcium phosphate (OCP: Ca8(HPO4)2(PO4)4·5H2O) are the main transient phases prior to brushite nucleation that occurs after ∼8 min. At pH > 11, hydroxyapatite (HAp: Ca10(PO4)6(OH)2) nucleates after >35 min, depending on the experimental conditions. The reaction mechanism steps for hydroxyapatite are more complex compared to brushite. For hydroxyapatite formation, amorphous calcium phosphate phases with different chemical composition (Ca(HPO4)1–x(PO4)(2/3)x·nH2O, with x in the range 0.2–1, and with different lifetime may form. Amorphous tricalcium phosphate (ATCP: Ca(PO4)2/3·nH2O or Ca3(PO4)2·nH2O when x = 1) is the most persistent phase which can either transform into OCP and then hydroxyapatite or directly evolve into hydroxyapatite at pH > 12.2. The presence of citric acid retards the transformation kinetics by increasing the nucleation times of brushite and hydroxyapatite but has little effect on the reaction mechanism steps. Finally, this study identifies new reactive pathways that characterize the formation of amorphous calcium phosphate phases and their transformation into brushite microcrystals or hydroxyapatite nanocrystals under abiotic conditions, relevant to a wide range of technological applications and natural environments.