Transformation From Amorphous Calcium Phosphate To Hydroxyapatite Biology Essay

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The process from the amorphous calcium phosphate phase to the hydroxyapatite (HAP) crystalline phase have been captured in a physiological fluid by a combination of in situ extinction detection and ex situ electronic microscopy, revealing the secret of the phase transformation and orientation controls during the initial stage of mineral formation.

The precipitation of calcium phosphate is not only an important process in biomineralization, but also an applicable method in phosphate recovery,1 which becomes increasingly important for the environment protection against the human activity associated with the phosphorus cycle.2 Increasing evidence dedicating that the amorphous calcium phosphate (ACP) phase plays a crucial role in the precipitation of calcium phosphate in a neutral solution and during biomineralization.3 Herein, the formation and evolution of ACP becomes an key issue in understanding and controlling of the precipitation of calcium phosphate minerals, among which hydroxyapatite (Ca10(PO4)6(OH)2, HAP) is thermodynamically the most stable phase in nature. Despite of the importance of ACP in biomineralization, the structure and behaviour of ACP is not well known. 'Posner's clusters', Ca9(PO4)6, are thought to be the building units in both synthesized and biological ACP, which provide the short range order information in the radial distribution function.4 However, the arrangements of Posner cluster and the inclusion of water, hydroxide and other ions in ACP are still unknown. ACP is an unstable phase, it will be transformed into more thermodynamically stable phase in solution. Although both synthetic and naturally formed HAP crystallites evolve from ACP, the final morphologies of the mineral products of HAP acquired by synthesis methods and from biomineralization are very different. The crystallites ranged in the spherulitic pattern are frequently found in the lab synthesis conditions,5 while the oriented packing of HAP crystallites are commonly found in teeth or bones.6 The aim of this work is to acquire the experimental evidence on how the final spherulites HAP occur from ACP.

Although the nucleation of crystallites from amorphous precursor phase is widely observed in varies systems, such as colloidal sphere,7 protein,8 and atomic crystallization, the detailed transformation pathway remains extensive debate.9 Varies pathways have been proposed, such as two-step8b, 10 or multi-step crystallization;7a nucleation inside amorphous phase7a or on the surface of amorphous phase.11 In the case of ACP transformation, the dissolution-reprecipitation12 and solution-mediated solid-solid transformation13 mechanisms have been suggested by Boskey, Posner, and Eanes et al. since the 1970s. Recent results showed that the crystallization of HAP from ACP may start at the inter-particles boundary,14 the ACP-solution interface,5b or inside the ACP.15 We note that the initial solution conditions (pH, ionic strength, ionic species and concentrations) in these works are quite different from the physiological solution (pH 7.4 buffered solutions, ionic strength 0.15 M), in which biomineralization takes place. In addition to pH and ionic strength, it has been reported that carbonate, magnesium, fluoride ions and some biomolecules in biological environment also have profound effects on the stability of ACP.4c, 16 Here, we take special attention on the formation and evolution of ACP under physiological conditions.

It is a great challenge to monitor the evolution of ACP in aqueous solution in situ, due to the instability of ACP and its fast transformation.13, 17 In this regard, we adopt an ex-situ approach in terms of fishing the samples by paddling the Formvar-Carbon coated copper grid from the reacting suspension. Owing to the hydrophobic carbon film, the progenitor solution can be blot off completely within only one second. The samples are further dried under lamp light, effectively terminating the transformation reaction. Herein, the morphology and phase evolution of the minerals can be examined by ex situ electron microscope (EM). Here we use UV/Visible microplate reader to study the mineralization process in situ, as there is abrupt changes in extinction of the ACP suspension as the phase transformation.18 (see in ESI†)

Fig. 1 The optical evolution of calcium phosphate solution. (a) Photographs of the calcium phosphate solution. The solution became turbid, then clear again companied with the sediment of minerals. (b) The turbid can be quantitatively determined by the extinction of the solution. The mineralization process can be divided into three stages.

After the mixing of calcium and phosphate solutions (with final concentration: 4 mM CaCl2, 6 mM K2HPO4, 150 mM NaCl, pH 7.40±0.05 buffered using 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), the saturation indices of initial solution were 0.75 (ACP, Ca3(PO4)2), 25.8 (HAP), 23.1 (HAP, after the precipitation of ACP) (see ESI), the solution became turbid gradually. At about 1 hour, the suspension suddenly became clear, companied with the visible sedimentations (Fig. 1a). The whole mineralization process can be monitored with the extinction curves (the optical difference of 405 nm and 550 nm) (in Fig. 1b). According to the extinction curve, the mineralization process can be divided into four stages (Fig. 1b). Briefly, in stage I, as the mixing of calcium and phosphate solutions, the ACP was formed. Due to the optical diffraction of the mineral, the suspension became turbid after mixing. In stage II, the extinctions of the solutions arise and then reach to a platform, resulting from the aggregation of ACP (Fig. 2a). In stage III, one will observe an abrupt drop of the extinction, corresponding to the transformation of ACP mineral (Fig. 2b, Figs. 2e-g).18 After the transformation, it follows stage IV, the crystallites underwent the further growth, ripen and aggregation. The final products were the spherulites, the pattern composed of platelet crystallites in radial orientation (Fig. 2c, i). The selected area diffraction (SAED) study indicated that each platelet in a spherulite was a single crystallite (Fig. 2c). The SAED and XRD patterns (Fig. S1†) of the solids matched those of HAP (JCPDS, 72-1243). Furthermore, FTIR spectrums confirmed the phase, and indicated the presence of carbonate in the minerals (which was caused by the dissolution of CO2 in air into the solutions) (Fig. S2†).19 The characteristic four stages in the extinction curves were also found under solutions with different calcium and phosphate concentrations (See ESI†, Fig. S3†), indicating that the amorphous transient phase commonly occur at the early stage during the precipitation of HAP.13, 17

The detailed morphology and phase evolution of ACP were captured by ex situ EM. The sphere-like minerals (215 nm ± 29 nm) were formed firstly (within 3 min, Figs. 2a, d). The SAED patterns of diffusive rings indicate that the spheres are amorphous (Fig. 2a). The initially round-shaped ACP particles indicate their fluid-like behavior at the very begining. The sintering of the submicro sized ACP droplets in solutions lead to the further aggregation of these spheres (Fig. 2a) rather than completly merged large sphere. Only a few partial merged ACP droplets were observed (Fig. 2d). The discrete ACP spheres (Fig. S4) were captured if the collagen-I was introduced in phosphate solutions at the very beginning (see details in ESI†), which may hindered the motion and aggregation of the submicro-spheres. After the formation of ACP, the particles remains in amorphous state for about 1 hour before the transformation. The transformation of ACP spheres were captured in detail: at first, the boundary of ACP spheres became polygonal-like (Fig. 2b, 2e, 3a; 67min). Some condensed ring or dots appeared in the SAED patterns (Fig. 2b), indicating that the crystallization. The bright dots in the dark field TEM images (DF-TEM) (Fig. 3b), indicated the occurance of crystallized minerals. The high resolution TEM images (HR-TEM) (Fig. 3c) directly show that the crystallization readily happened at the surface of the ACP sphere, while the main portion of such a sphere was still amorphous inside (ie. FFT patterns in Fig. 3c, region 3).The initial formed crystallites were about 3-5 nm, having several crystalgraphic units of HAP in depth (Fig. 3c, region 1).

Fig. 2 Phase and morphology evolution of calcium phosphate minerals. (a-c) TEM images and SAED patterns of the precipitates. (a) Initial formed ACP spheres. Bar: 1 mm and 100 nm (subimage). (b) Intermediate state of ACP. Bar: 1 mm and 100 nm (subimage). (c) Final spherulite HAP. Bar: 1 mm and 200 nm (subimage). (d-i) SEM images of the evolution of ACP. (d) 3 min. (e-g) 67-73 min. (h) 90 min. (i) 7 hr. Bar: (d-f) 100 nm; (g) 200 nm; (h-i) 1 mm.

Fig. 3 Initial phase transformation stage of ACP particles. (a) SEM image (b) DF-TEM image. (c) HR-TEM image, and the FFT patterns of crystallized regions (1, 2) and the amorphous region (3). Bar: (a) 100 nm; (b) 50 nm; (c) 10 nm.

The initial phase of the crystallites are determined by the HR-TEM. According to the FFT patterns of the HR-TEM images (Fig. 3c, region 1, 2), the d space of the lattice is about 0.81 nm, confirming that the initial formed crystal phase is HAP rather than octacalcium phosphate (OCP) (in which d space was 1.87 nm, JCPDS 79-0423). The lattice images showed that the {100} faces, the most stable face of HAP, were preferred to be exposed to the ACP-solution interfaces (Fig. 3c, region 1, 2), which may be caused by the lower free energy for the heterogeneous nucleation at the ACP-solution interface than that of homogenous/heterogeneous nucleation in bulk solution or inside ACP phase.

Afer the trigger of crystalization, some mineral fingers were found to stick out of the surface of ACP spheres just in a few minutes, indicating the fast radial growth of the minerals (Fig. 2f). As the result of the overgrowth of the crystallites on the surface of ACP spheres (Fig. 2e-g, 67-73 min), the minerals were covered by a layer of crystallites (Fig. 2g) within 10 min. At this stage, the morphology of the minerals changed dramatically from compact sphere-like (Fig. 2e) to a spherulitic pattern (Fig. 2g). Note that it only took less than 10 minutes for the minerals to grow from about 200 nm to 1 mm (Figs. 2e-g). In contrast, no obvious growth of ACP was found before the crystallization (Figs. 2d-e; take about 1 hour). The radial growth of the crystallites on the ACP surface, and the preferential growth of crystallites along the <100> and <001> directions of HAP together resulted into the spherulite HAP (Fig. 2h; 90 min.). The subunits of the aggregates were the platelet HAP. Further incubation of the minerals lead to the formaiton of larger spherulites and the platelets became more obvious (Fig. 2c, i; 7hr). The angular relationship between the SAED diffraction dots (Fig. 2c) of the platelet further confirmed that the final phase was biological-like apatite.20 (see in Figure S5†, Table S1†)

In summary, by combing the in situ detection of the extinction of suspensions and ex situ EM observation of minerals, both the mineralization process and the detailed phase and morphology evolution from ACP spheres to the HAP spherulites have been captured. At the initial stage of the transformation, the nucleation occurs preferably at the surface of ACP spheres. The results clearly show that the random orientations of the crystal seeds on ACP and the subsequence radial growth of HAP is the mechanism for the formation of the spherulitic crystallites. The direct evidence for the initial transformation of ACP to HAP advances the fundamental understanding on the amorphous phase mediated HAP crystallization. We find that once the onset of crystallites, they will grow along the initial sitting directions. The embedding/adhering of the crystal seeds on ACP surface would not allow the crystallites to rotate their orientations and dislocate their relative positions. The geometrical selection gives rise to the final spherulitic morphology. In this regard, the oriented nucleation of ACP in biomatrix space would be the key issue in ordered biomineralization.

This work was supported by Supported by the Singapore ARF Project No. T206B1114, and National Natural Science Foundation of China (20701032 and 20871102) and the MOST of China Grant No. 50928301.

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