Ruiz-Agudo Encarnacion, Ruiz-Agudo Cristina, Di Lorenzo Fulvio, Alvarez-Lloret Pedro, Ibañez-Velasco Aurelia, Rodriguez-Navarro Carlos
Department of Mineralogy and Petrology, University of Granada, Fuentenueva s/n, Granada 18071, Spain.
Physical Chemistry, Department of Chemistry, University of Konstanz, Universitätsstraße 10, Konstanz 78457, Germany.
ACS Biomater Sci Eng. 2021 Jun 14;7(6):2346-2357. doi: 10.1021/acsbiomaterials.1c00196. Epub 2021 May 11.
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 axis. Such nanoplates are made up of HAp nanocrystallites with a preferred 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.
羟基磷灰石（HAp）是骨骼中的主要无机相，其矿化遵循非经典结晶途径，涉及亚稳前体，并且受到有机大分子的强烈影响。然而，柠檬酸盐等小分子对HAp形成的影响尚未得到充分的限制。通过电位滴定实验和滴定热分析，并结合多分析方法，我们表明柠檬酸盐稳定了预成核物种以及在系统中任何固相成核之前形成的类液状磷酸钙前体。类液状前体相的稳定化可能有助于在骨矿化过程中渗透到胶原纤维的腔中，这解释了先前研究中报道的柠檬酸盐对胶原介导矿化的增强作用。因此，柠檬酸盐可以在任何固相（无定形或结晶）形成之前影响骨矿化。我们还表明，在不存在和存在柠檬酸盐的情况下，无定形磷酸钙（ACP）之后形成的HAp会产生约5-12nm厚的纳米片，沿c轴拉长。这种纳米片由具有优选c轴取向且散布有ACP的HAp纳米微晶组成。纳米片的形态、尺寸和优选的晶体学取向与骨HAp纳米晶体非常相似，似乎是由无定形前体形成的HAp的固有特征。我们的结果挑战了目前关于骨中HAp矿化的模型以及柠檬酸盐的作用，为回答长期存在的问题——为什么自然进化青睐HAp作为骨中的矿相——提供了新的线索。
