Citrate Stabilizes Hydroxylapatite Precursors: Implications for Bone Mineralization.

Encarnacion Ruiz-Agudo,Pedro Alvarez-Lloret,Fulvio Di Lorenzo,Aurelia Ibañez-Velasco,Cristina Ruiz-Agudo,Carlos Rodriguez-Navarro

doi:10.1021/acsbiomaterials.1c00196

Abstract

Mineralization of hydroxylapatite (HAp), the main inorganic phase in bone, follows nonclassical crystallization routes involving metastable precursors and is strongly influenced by organic macromolecules. However, the effect of small organic molecules such as citrate on the formation of HAp is not well constrained. Using potentiometric titration experiments and titration calorimetry, in combination with a multianalytical approach, we show that citrate stabilizes prenucleation species as well as a liquid-like calcium phosphate precursor formed before any solid phase nucleates in the system. The stabilization of a liquid-like precursor phase could facilitate infiltration into the cavities of the collagen fibrils during bone mineralization, explaining the enhancement of collagen-mediated mineralization by citrate reported in previous studies. Hence, citrate can influence bone mineralization way before any solid phase (amorphous or crystalline) is formed. We also show that HAp formation after amorphous calcium phosphate (ACP) in the absence and presence of citrate results in nanoplates of about 5–12 nm thick, elongated along the c axis. Such nanoplates are made up of HAp nanocrystallites with a preferred c axis orientation and with interspersed ACP. The nanoplatelet morphology, size, and preferred crystallographic orientation, remarkably similar to those of bone HAp nanocrystals, appear to be an intrinsic feature of HAp formed from an amorphous precursor. Our results challenge current models for HAp mineralization in bone and the role of citrate, offering new clues to help answer the long-standing question as to why natural evolution favored HAp as the mineral phase in bone.

Highlights

Many living organisms take advantage of nonclassical crystallization routes to build complex hierarchical mineral microstructures.[1−3] Defective hydroxylapatite (Ca10(PO4)6(OH)[2]; HAp), or bioapatite, the biomineral composing mammalian bone and teeth, is one of the most prominent examples
We show how a kinetically induced dense liquid precursor is formed prior to HAp crystallization and amorphous calcium phosphate (ACP) nucleation during calcium phosphate formation, which is stabilized in the presence of citrate
Our findings provide a mechanism that explains earlier claims that citrate improves collagen-mediated mineralization by calcium phosphate, a role that has been previously assigned to macromolecules (NCPs)

Summary

Introduction

Many living organisms take advantage of nonclassical crystallization routes (e.g., formation of amorphous precursors or growth by the nanoparticle assembly) to build complex hierarchical mineral microstructures.[1−3] Defective hydroxylapatite (Ca10(PO4)6(OH)[2]; HAp), or bioapatite, the biomineral composing mammalian bone and teeth, is one of the most prominent examples. It is long known that HAp biomineralization entails the formation of intermediate states such as amorphous precursors,[4] the exact conversion mechanism to crystalline HAp is still under debate. It has been proposed that prenucleation clusters are involved in HAp formation.[5−7] These prenucleation species could be considered as Posner’s clusters, first postulated in 1975,8 and later found in simulated body fluids using an intensityenhanced dynamic light scattering (DLS) technique.[9] These clusters were envisaged as the solution precursors to amorphous calcium phosphate (ACP).[9] In addition, the assumed fluidic character of calcium phosphate precursors is critical for achieving optimal collagen mineralization.[2,10] despite the significant experimental evidence of “nonclassical” processes during bone biomineralization, the exact steps behind the biological HAp formation are largely unknown

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