The Characterization of Hydroxyapatite and Octa-calcium Phosphate with Electron Energy Loss Spectroscopy

Hydroxyapatite (HA, Ca10(PO4)6(OH)2) is the major mineral component in most organisms. The study of the formation of bones and teeth revolves around the phase transformation of HA [1–3]. Octacalcium phosphate (OCP, Ca8H2(PO4)6) is considered a metastable phase and the precursor for the formation of HA [4–6]. In 2018, Simon et al. observed the presence of OCP binding with osteocalcin in the formation of bone [7] Exposing phase transformation from OCP to HA would largely help the understanding of mineral formation in animals, and further help the design and repairment of artificial bones and teeth. However, the detection and differentiation of OCP and HA remain challenging because of their similar compositions and crystal structures. In 2018, a large portion of OCP was detected in the matrix metalloproteinase-20 (MMP20) knocked-out teeth by Yamazaki with Raman microspectroscopy [8]. Recently, our group discovered that the hypomineralized teeth induced by the lack of MMP20, and the major component of the enamel crystals was similar to OCP instead of HA based on the selected area diffraction pattern (SADP) images [9]. However, with Raman microspectroscopy, the detection of OCP lacks spatial information. With the SADP images, although we verified the crystal structure of the hypomineralized enamel crystals was consistent with the one from OCP, it was not clear whether the crystals were directly from OCP. In this research, electron energy loss spectroscopy (EELS) analysis was used to distinguish the HA and OCP crystals. EELS provides not only information on chemical bonding but also quantification of chemical composition. In addition, STEM- EELS offers an improved spatial resolution. Commercial HA and synthesized OCP were first characterized as the reference materials for the EELS analysis. As shown in Figure 1A, the synthesized OCP was initially impure, but after heating in distilled water at 80°C for 16 hours, the impure minerals transformed into OCP. The reference HA and OCP could be easily distinguished by the difference in microstructures (Figure 1B). For the EELS results from the Phosphorus-L2,3 and Calcium-L2,3 edges, the spectrums were similar (Figure 1C and D). The shoulder peaks at 142 eV were regarded as the transition to the empty orbitals in calcium, indicating the detected phosphate groups bonded to calcium [10]. In the Oxygen-K edge spectrums (Figure 1E), a slight difference was observed between the two spectrums. Aside from the difference in the energy loss near-edge structure, the EELS quantification of the Ca/P ratio for the sphere and the plate particles was approximately 1.6 and 1.3, respectively. The quantification results were close to the Ca/P ratios of HA and OCP, which are 1.66 and 1.33, respectively. Based on this difference, the minerals of immature mouse teeth were characterized. The TEM specimens from wild-type (WT) mice were prepared by focused ion beam-scanning electron microscopy (FIB-SEM). The bright field TEM images and the EELS from corresponding regions were shown in Figure 2. For the P-L2,3 and Ca-L2,3 edges, except for the spectrum from the spaces of Weber (SW), all spectrums were similar and have the shoulder peak at 142 eV, indicating the lack of P and Ca in the SW region and the presence of calcium phosphate components in the other regions. In the O-K edge, the spectrums from dentin and enamel were similar to the two-peak featured HA spectrum, and the one from SW was too noisy to identify. In this work, the reference EELS of HA and OCP is established and discussed. Based on these results, the minerals in the immature mouse teeth were identified as HA instead of OCP. The quantification study with additional biological specimens will be carried out in the future. This research can not only help to further understand the phase transformation and formation of teeth, but also explore the potential of EELS as a great tool for dental material identification. (A) X-ray diffraction (XRD) patterns of the synthesized OCP with and without post treatments. The peaks labeled red stars fit the pattern of dicalcium phosphate dihydrate and the ones labeled red circle fit the pattern of HA. (B) TEM bright field images: the spheres are HA; the plate is OCP. (C to E) The EELS results from phosphorus-L2,3 (C), calcium-L2,3 (D), and oxygen-K edge (E). (A and B) Bright field TEM images from the wild-type (WT) immature mouse incisor. (C to E) EELS results of the phosphorus-L2,3 (C), calcium-L2,3 (D), and oxygen-K edge (E) from the corresponding regions labeled in (A) and (B).

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