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Calcium phosphate precipitation in hanging drop vapor diffusion monitored by in situ Raman microspectroscopy

Gloria Belén Ramírez-Rodríguez ,  José Manuel Delgado López ,  Jaime Gómez-Morales 

Instituto Andaluz de Ciencias de la Tierra (LEC-IACT), Avda. Las Palmeras, nº 4. Armilla, Granada 18100, Spain

Abstract

Calcium orthophosphates have a special interest in biomineralization and biomedical fields because of their presence in mineralized tissues of mammals and their wide variety of applications [1] ranging from biomimetic bone graft and tissue regeneration to carrier matrices of drugs and proteins. Among them, hydroxyapatite (Ca5(PO4)3(OH), HA), the thermodynamically most stable under physiological conditions, represents a model compound of the inorganic constituent of bones, teeth and many pathological calcifications [2]. Bone apatites are non-stoichiometric calcium and OH deficient nanosized crystals doped with carbonate (4–6 w/w%) and different foreign ions such as Na (0.9%) and Mg (0.5%).[2] Other phases such as dicalcium phosphate dihydrate (CaHPO4.2H2O, brushite, DCPD) and octacalcium phosphate (Ca8H2(PO4)6.5H2O, OCP) are usually found in more acidic solutions [3]. In recent years, a huge number of studies on the crystallization process of these compounds have been carried out in order to analyse the mechanisms of precipitation and stability of each calcium phosphate (CaP) phase [4]. DCPD and OCP have been suggested as possible metastable precursors in the formation of apatite. This may occur by the precipitation of DCDP and/or OCP followed by its transformation to a more apatite phase.

We have studied the evolution of calcium phosphate precipitation by hanging drop vapor diffusion  using the “crystallization mushroom” [5]. The hanged drops initially contained mixed solutions of Ca(CH3COO)2 and (NH4)2HPO4. The diffusion of CO2 and NH3 gases released from NH4HCO3 solutions (located in the lower chamber of the device) at different concentrations (30 mM, 100 mM and 2 M) produced a gradual increase of the drop pH. The pH increase rate as well as the final pH strongly depends on the NH4HCO3 concentration. The evolution of the calcium phosphate precipitation was followed in-situ by confocal Raman microspectroscopy.Time-dependent in situ Raman spectra indicated that amorphous calcium phosphate (ACP) was the first precipitate appearing just after mixing the Ca- and PO4-containing solutions. It transformed to dicalcium phosphate dihydrate (DCPD) a few minutes later. The lifetime of DCPD strongly depends on the concentration of the NH4HCO3 solutions and thus on the pH increase rate. The pathway for the phase transformation from ACP to DCPD and then to octacalcium phosphate (OCP) followed a dissolution–reprecipitation mechanism. The precipitates obtained after 7 days of vapor diffusion also depends on the NH4HCO3 concentration. At lower concentrations (30 and 100 mM), OCP, micrometric HA and nanosized HA was obtained. OCP played the role as temporal template for the heterogeneous nucleation and crystallization of biomimetic carbonate–apatite nanocrystals. In contrast, working with the highest NH4HCO3 concentration the system evolved to the precipitation of elongated calcite crystals.

Acknowledgements. Financial support from Spanish MINECO (Projects MAT2011-28543 and Consolider-Ingenio 2010, Factoría de Cristalización) and CEI-BioTic (UGR) is greatly acknowledged.

References

[1] Dorozhkin S. Calcium Orthophosphates in Nature, Biology and Medicine. Materials 2009;2:399-498.

[2] Mann S. Biomineralization: principles and concepts in bioinorganic materials chemistry. Oxford: University Press; 2001.

[3] Johnsson MS-A, Nancollas GH. The Role of Brushite and Octacalcium Phosphate in Apatite Formation. Crit Rev Oral Biol Med 1992;3:61-82.

[4] Gómez-Morales J, Iafisco M, Delgado-López JM, Sarda S, Drouet C. Progress on the preparation of nanocrystalline apatites and surface characterization: Overview of fundamental and applied aspects. Prog Cryst Growth Ch 2013;59:1-46.

[5] Ramirez-Rodriguez GB, Delgado-Lopez JM, Gomez-Morales J. Evolution of calcium phosphate precipitation in hanging drop vapor diffusion by in situ Raman microspectroscopy. CrystEngComm 2013;15:2206-12.

 

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Presentation: Oral at 17th International Conference on Crystal Growth and Epitaxy - ICCGE-17, General Session 3, by José Manuel Delgado López
See On-line Journal of 17th International Conference on Crystal Growth and Epitaxy - ICCGE-17

Submitted: 2013-04-15 19:10
Revised:   2013-07-16 16:49