The Kinetics And Mechanisms For Phase Transformation At The Calcium Phosphates-Solution Interface

As one of the most widely studied inorganic compounds,calcium phosphate（Ca-P）minerals are widely present in the earth’s lithosphere（e.g.,phosphate ores）and organisms（e.g.,teeth,bones,antlers,and partial pathological calcifications）.Whether in the soil environment or in organisms,there are a variety of Ca-P metastable phases with high solubility that eventually gradually transform into the most thermodynamically stable phase(hydroxyapatite,HAP,Ca₁₀（PO₄）₆（OH）₂).Nonetheless,probing the phase transition process of various metastable phases to HAP at the microscale remains unknown.In addition,the Kinetics of specific reactions at the mineral-water interface are largely regulated by the various organic/inorganic molecules/ions contained in the solution phase at the interface.Therefore,in this study,acidic amorphous calcium phosphate（ACP）,calcium hydrogen phosphate dihydrate（DCPD）,and calcium hydrogen phosphate monohydrate（DCPM）were selected as the research objects,and by a combination of in situ the Atomic Force Microscopy（AFM）,Raman Spectroscopy and High-Resolution Transmission Electron Microscope（HRTEM）,we explore the kinetics and mechanism of the phase transition reaction of these metastable phases at the mineral-water interface under the action of organic and inorganic additive.The exploration of this micromechanism can provide mechanistic insights for understanding the cycling of soil mineral elements at the mineral-water interface,the bioavailability of phosphorus nutrients,and the formation of calcium phosphate minerals in organisms.The main results from this study are summarized below:（1）Using the phosphorylated/unphosphorylated Ser-Ser-Glu-Glu-Leu（SSEEL）fragment within Amelotin（AMTN）as a model template revealed the role of phosphorylated/unphosphorylated fragment to the phase transformation of acidic amorphous calcium phosphate（ACP）to hydroxyapatite（HAP）.We used in situ AFM and HRTEM to observe the regulation of the phosphorylated/non-phosphorylated SSEEL fragment on the phase transition of acidic ACP to HAP.The（p）S（p）SEEL motif with highly negative charges could form a protection layer to stabilize the acidic ACP particles to inhibit HAP crystal formation.Meanwhile,（p）S（p）SEEL peptides can bind to Ca²⁺on the acidic ACP surface,attracting more Ca²⁺to the surface from its interior,resulting in a local high Ca²⁺microenvironment.This promoted the formation of another amorphous calcium phosphate（named as ACP-II）different from the parent phase.In addition,due to the local high-calcium environment around ACP-II,it first phase transformed into a more stable octacalcium phosphate（OCP,Ca₈H₂（PO₄）₆·5H₂O）,which became the initial phase transformation site.By contrast,the nonphosphorylated SSEEL motif strongly complexes and chelates Ca²⁺ions from the acidic ACP surface to promote the dissolution and subsequent reprecipitation of HAP.In addition,the active non-phosphorylated SSEEL peptide may contain secondary structures that nduce HAP to grow into needle-like crystals along the C-axis.This result is helpful for understanding the regulatory mechanism of phosphorylated/non-phosphorylated proteins on the transition from amorphous to crystalline phase.（2）In situ probed the phase transformation mechanism of DCPD to HAP and using the acidic Asp（D）-rich osteopontin（OPN）fragment（DDVDDTDDSHQSDE）as a model protein revealed theregulation mechanism of acidic proteins to the above phase transformation.We used in situ AFM and Raman spectroscopy to determine the phase transformation of DCPD to HAP.The phase transformation of DCPD to HAP through two processes.First,dissolution of DCPD results in the reprecipitation of an acidic ACP.These amorphous phases preferentially nucleate on the[（?）00]_Cc step edges of DCPD and then gradually expand to the entire crystal plane.Subsequently,this acidic ACP absorbs excess calcium ions in the environment and gradually phase-transforms into HAP crystals.Furthermore,in the presence of acidic OPN peptide segments,it can inhibit the phase transformation of DCPD to HAP by preferentially absorbing the edges of[（?）00]_Cc step edges to reduce the nucleation sites of acidic ACP.The findings provide a methodological extension for exploring the solid phase mineral replacement reaction process at the mineral-water interface.（3）DCPM can rapidly transform into a more stable octacalcium phosphate（OCP）at the mineral-water interface,and in the the presence of magnesium ions(Mg²⁺)can inhibit the formation and phase transformation process of DCPM.As a newly discovered calcium phosphate crystal phase in 2020,DCPM has not been fully studied yet.We firstly prepared micrometer-sized DCPM from supersaturated DCPD solution-methanol mixture.Using in situ AFM and Raman spectroscopy to determine,we found acidic ACP is first generated in this mixed system and then converted into DCPM by dissolution-reprecipitation,and Mg²⁺can inhibit the formation of DCPM by reducing the solubility of the acidic ACP.Furthermore,we confirmed that DCPM has higher solubility than other Ca P crystalline phases,which can rapidly transform into more stable OCP at the mineral-water interface,however Mg²⁺can suppress the above-mentioned phase transformation process.This achievement provides the possibility for the application of DCPM in the future.