Insights for OCP Identification and Quantification in the Context of Apatite Biomineralization
Calcium phosphates (CaPs) are one of the major mineral families of wide interest in biomineralization and biomaterials development. The identification and quantification of the different CaPs phases (crystalline and amorphous) remains a major challenge in both in vitro and in vivo systems. This work aims to provide critical analysis of the different characterization techniques, Raman spectroscopy, solid-state NMR spectroscopy (ssNMR) and X-ray diffraction (XRD) that are used for the discrimination of octacalcium phosphate (OCP) from hydroxyapatite (HAp). Although remaining not suitable for quantification purpose, Raman allows the detection of quite low amount of OCP (~10 wt%) in presence of biomimetic carbonated apatite (cHAp), providing that the (HPO4) contribution of weak intensity from OCP at 1008 cm-1 is observable or that spectral decomposition is performed. At reverse, the quantification of OCP in proportions down to 10 wt% is easily performed with 31P solid state NMR. Opposite to solid state NMR, Raman can be implemented under in situ conditions to follow the time dependence of the biomineralization process without any sampling perturbation. An approach combining the advantage of in situ micro-Raman spectroscopy and the sensitivity of ex situ solid state NMR was used to monitor the formation of biomimetic carbonated apatite from an acidic aqueous solution of phosphate, carbonate and calcium ions. In relationship with the progressive increase of pH, we identify the transient phases, precursors of cHAp: it is shown that an amorphous calcium phosphate phase (ACP) is first formed and subsequently transformed into OCP that then progressively turns into cHAp. Finally, powder X-ray diffraction coupled to Rietveld refinement was found very powerful to quantify very few amount of residual OCP in cHAp (down to 2 wt%). Upon the OCP transformation into cHAp, the decrease of the relative intensity of the (100) diffraction peak of the transient OCP phase was ascribed to an alteration of its hydrated layers, related to an incorporation of water molecules and/or carbonate anions upon reaction time. Such carbonate uptake in the course of transformation of OCP into cHAp could explain the origin of carbonate substitutions in the final biomimetic carbonated apatite.