Experimental evidence and structural modeling of nonstoichiometric (0 1 0) surfaces coexisting in hydroxyapatite nano-crystals

https://doi.org/10.1016/j.colsurfb.2011.08.016Get rights and content

Abstract

High-resolution transmission electron microscopy (HRTEM) and ab initio quantum-mechanical calculations of electronic structure were combined to investigate the structure of the hydroxyapatite (HA) (0 1 0) surface, which plays an important role in HA interactions with biological media. HA was synthesized by in vitro precipitation at 37 °C. HRTEM images revealed thin elongated rod nanoparticles with preferential growth along the [0 0 1] direction and terminations parallel to the (0 1 0) plane. The focal series reconstruction (FSR) technique was applied to develop an atomic-scale structural model of the high-resolution images. The HRTEM simulations identified the coexistence of two structurally distinct terminations for (0 1 0) surfaces: a rather flat Ca(II)-terminated surface and a zig-zag structure with open OH channels. Density functional theory (DFT) was applied in a periodic slab plane-wave pseudopotential approach to refine details of atomic coordination and bond lengths of Ca(I) and Ca(II) sites in hydrated HA (0 1 0) surfaces, starting from the HRTEM model.

Highlights

HRTEM crystal simulation reveal the coexistence of two HA (0 1 0) terminations. ► A Ca-rich (B layer) and PO4-rich (A layer) were identified. ► The HA nano-crystal along [1 0 0] is formed by integer numbers of the BAA sequence. ► Water molecules are differently adsorbed on each crystal surface.

Introduction

Hydroxyapatite (HA), Ca10(PO4)6(OH)2, is a widely used biomaterial for bone and tooth regeneration due to its biocompatibility, its ability to associate with molecules, ions and metals and its osteoconductivity [1], [2], [3]. A major challenge of current research is to produce apatite-based materials with physico-chemical and morphological characteristics that stimulate osteogenesis when implanted in an injured region [4], [5], [6], [7]. To succeed in this effort it is necessary to understand the mechanisms that regulate the interaction of HA surfaces with biomolecules and cells leading to biomineralization and the formation of hard tissues [8], [9], [10], [11], [12]. To this end, the physico-chemical properties of HA surfaces such as structure, stoichiometry, solubility, crystallinity, charge, surface energy and topography must be determined in the micro and nano-scale [13], [14], [15], [16], [17], [18], [19]. The present view of biomineralization processes considers that every biogenic mineral has a precursor phase that can be amorphous and/or composed of a different mineral phase. In the case of apatite from bone, it has been found that small round particles of ∼1–2 nm in diameter are present in association with type I collagen molecules and agglomerated small round particles are present in direct association with early nanocrystal formation [20]. The low misorientation angle between components of apatite crystals in different biological models [21], [22] indicates that the nanometer-sized particles were nucleated, shape modulated and oriented under the influence of molecules, in agreement with the non-classical crystallization mechanism [23]. The above hypotheses suggest that the early formation of hydroxyapatite is strongly and simultaneously dependent on surface energy and the interaction with the substrate so that the small nuclei can orient approximately in the same direction [20], [21], [22], [23].

In recent years, the focus of much experimental and theoretical research based on calcium phosphates has shifted to the HA surface. In this subject, many advances have been made on the structural characterization of HA surfaces and their interactions with metals and molecules [24], [25], [26], [27]. From the experimental point of view, high resolution transmission electron microscopy (HRTEM) became one of the most powerful characterization techniques of the ultra-structure of HA nanoparticles precipitated under ex vivo and in vivo conditions [28], [29], [30], [31] because of its ∼1 Å resolution. Although this technique has been extensively used, experimental limitations appear when very small crystals are analyzed. One of the most critical effects is the electron beam damage on HA nanocrystals. Long-term exposure to an electron beam may change the surface structure and particle morphology, induce crystallization and produce amorphization.

The hydroxyapatite nanocrystals produced by wet precipitation have a preferential growth along the [0 0 1] direction with large (0 1 0) surfaces that dominate apatite interactions with the biological medium. Previous HRTEM spectroscopy measurements carried out by Sakhno et al. [32] characterized the morphology of HA nanoparticles surfaces. Amorphous and crystalline surfaces were observed, resolving (0 1 0), (1 0 0) and (0 0 1) crystalline surface planes. Infrared spectroscopy was used to investigate surface hydration properties, surface hydroxyl groups and CO adsorption. Strong similarities between amorphous and crystalline surfaces were observed. Sato et al. [33] conducted a detailed HRTEM study effects of an electron beam on sintered HA structure and characterized the crystalline–amorphous interfaces and the grain boundaries. They showed that the HA (0 1 0) crystal structure is terminated by a plane crossing the hydroxyl columns on which Ca2+ sites and PO43− tetrahedra are located.

Meticulous theoretical investigations have been carried out on the electro-neutral stoichiometric and non-stoichiometric (Ca-rich and PO4-rich) terminations accessible in the (0 1 0) HA surface [25], [26], [27]. These studies focused on energetic stability, surface characterization from electrostatic potential maps and adsorption mechanisms of low (up to two) and higher loadings (five) of H2O molecules on Ca and PO4 sites. However, information on structural environment and water adsorption considering the two non-equivalent Ca(I) and Ca(II) sites separately, much-discussed in HA bulk [34], [35], has been detailed only for the (0 0 1) HA surface [24].

Although the Ca-rich termination has been identified by Sato et al., the PO4-rich and the stoichiometric (0 1 0) terminations were not yet detected by HRTEM. These results highlight the relevance of new experimental evidence presented here, to improve our understanding concerning the formation and chemical reactivity of HA nanocrystal (0 1 0) facets.

In the present work, the HRTEM technique was used to analyze surface profiles of hydroxyapatite nanoparticles precipitated from aqueous solution at 37 °C with preferential growth along the [0 0 1] direction and large (0 1 0) faces. The high-resolution images were interpreted by using an HA nanocrystal model generated using the MEGACELL software [36] for HRTEM multislice simulations [37]. The simulated nanocrystal was used to investigate the two different (0 1 0) faces observed in a single nanoparticle of 50 nm length and 8.49 nm width. This analysis permitted the characterization of the non-stoichiometric HA surface terminations [25], [26]. Starting from the HRTEM results, periodic density functional theory (DFT) was used to model and refine hydrated (0 1 0) HA surfaces with the two different terminations in order to obtain information about the structural modification and chemical environment around the Ca(I) and Ca(II) sites.

Section snippets

Sample preparation

Hydroxyapatite nanoparticles were precipitated using a wet chemical method at 37 °C by the addition of diammonium phosphate ((NH4)2HPO4, Merck) to a calcium nitrate tetrahydrate (Ca(NO3)2·4H2O, Merck) aqueous solution, under slow magnetic stirring and a pH of 11. For compatibility with a physiological state, the precipitation temperature was set at 37 °C. After addition of reagents, the precipitate was aged for 2 h in order to improve crystallization. Afterwards, the solid, labeled as HA37, was

XRD and FTIR

X-ray powder diffraction pattern of sample HA37 showed the existence of a single crystalline phase characterized as hydroxyapatite, as seen in Fig. 2. The FTIR spectrum, not shown, confirmed this attribution by the presence of OH vibrational modes at 3470 cm−1 and 634 cm−1 and PO43− vibrational modes ν2 (470 cm−1), ν3 (962, 1037 and 1091 cm−1) and ν4 (603 and 565 cm−1). Vibrational modes at 1640 and 3448 cm−1 were attributed to structural and adsorbed water, respectively. Impurities of CO3 ions in PO4

Conclusions

In this work HRTEM was used to characterize hydroxyapatite elongated rod nanoparticles with preferential growth along the c direction. HRTEM crystal simulated images reveal the coexistence of two (0 1 0) terminations: Ca-rich (BA layers, zig-zag structure) and PO4-rich (A layer, flat structure). The stacking of BAA sets assures the crystal stoichiometry along the [0 1 0] direction. Our finding on the differences between the two (0 1 0) crystal faces may be related to the fine tuning of the apatite

Acknowledgements

The authors thank the National Council for Scientific and Technological Development (CNPq) Brazilian agency for the financial support no. 142892/2005-0; the Electron Microscopy Laboratory at the Brazilian Nanotechnology National Laboratory and the Brazilian Synchrotron Light National Laboratory for the technical support during electron microscopy and powder X-ray diffraction work respectively; the US Department of Energy through the Institute for Catalysis in Energy Processes at Northwestern

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