Citrate regulates extracellular matrix mineralization during osteoblast differentiation in vitro

Highlights

• A new osteoblast mineralization experiment model was proposed.

• Citrate stabilizes dicalcium phosphate dihydrate and octacalcium phosphate like precursors.

• Citrate inhibits the transformation of two precursors into hydroxyapatite.

• Citrate results in the smaller size and lower crystallinity minerals.

Abstract

The extremely high levels of citrate in bone highlight its important role, which must be involved in some essential functional or structural role that is required for the development and maintenance of normal bone. However, biomineralization researches have emphasized the interaction between the citrate and inorganic minerals during crystallization in cell-free systems. It is difficult to obtain a thorough and comprehensive understanding from cell-free experimental conditions and treatment methods. In this study, by proposing an osteoblast mineralization experimental model, we explored the regulation of citrate on bone apatite crystal structure. Our studies show that citrate stabilizes two precursors and then inhibits their transformation into hydroxyapatite. Concomitantly, the smaller size and lower crystallinity mineral deposition emerge during citrate-mediated osteogenic mineralization. These findings may provide a new perspective for the mechanism of osteogenic mineralization and a basis for further understanding of bone metabolism.

Introduction

Bone contains large amounts of citrate, which consists of ~1.6% of the bone content, ~5% of the organic component of bone [1]. Up to 80% of the total citrate in the body resides in bone [2,3]. In the past 35 years, clinical and biomedical researchers have neglected the meaning of citrate in bone and it has not even been described as a component of bone in recent textbooks and reviews [4,5]. Recent studies have found the binding relationship of citrate in the structure of apatite nanocrystals [4,[6], [7], [8], [9]]. It has been shown that hydroxyapatite (HAP) formed thinner nanocrystals in the presence of citrate in vitro [[10], [11], [12]]. The citrate production in osteoblasts may be an important source of citrate in bone [2,3]. These convincing evidences show that citrate is essential for normal bone formation that exhibits the important properties of stability, strength and resistance to fracture. Therefore, citrate is widely used as a drug for osteoporosis, and it has been proved that the application of citrate can effectively lead to an increase in bone density [[13], [14], [15], [16]].

It is generally accepted that the discovery of the role of citrate in collagen mineralization is of critical importance to improve bone generation and the treatment of bone-related diseases. Unfortunately, this important issue has not been well elucidated, and there are still intensive debates [4,8,9,17]. Many previous studies have focused on the interaction among citrate molecules, HAP nanocrystals and final mineral phase by analyzing the inorganic phase structure of bone tissue, and concluded that the binding of citrate on the HAP surface affects the mineral morphology and size [4,8,9]. However, biomineralization researches have emphasized the interaction between the citrate and inorganic minerals during crystallization in cell-free systems [[17], [18], [19]].

Two different ways of biomineralization have been explored through in vitro cell-free experiments. On the one hand, collagen fibrils were mineralized in vitro without any organic/polymer additives and the amino acid side chain of collagen peptide might provide the mineralized binding sites for calcium and phosphate ions (supersaturated solution) [20,21]. On the other hand, many in vitro experiments were carried out in the presence of non-collagenous proteins or peptides rich in polycarboxylic acids to prove their role in controlling mineral nucleation and growth [19]. It is worth noting that once the calcium and phosphate ion concentration exceeds the solubility product of the relevant phase in the extracellular environment, calcium phosphate particles are generated [22,23]. It should be explained that whether the process is mediated by osteoblasts and, if so, how the citrate regulates the size and morphology of apatite nanoparticles in a cell system model.

In this study, we focused on in vitro osteoblast mineralization experiment model, aimed at gaining a better understanding of the role of citrate in bone mineral formation and monitoring the transformation of mineral phases during osteoblast differentiation in vitro. By combined analyses of the high resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD), we have indicated that the origin of HAP from the precursor phases during osteoblast differentiation and the role of citrate in stabilizing amorphous precursor at the early stage and controlling nanocrystals size.