The accuracy of quantifying the degree of hard tissue calcification using an electron probe micro analyzer, micro-focus X-ray computed tomography, and tissue sectioning methods

Abstract

Objectives: Micro-focus X-ray computed tomography (μCT) helps evaluate specimens without destroying it. However, its accuracy of quantifying bone mineral density remains to be fully elucidated. We aimed to verify the accuracy of calcification assessed by μCT, by comparing the images of identical specimens obtained via different methods such as μCT and electron probe micro analyzer (EPMA) analyses.

Methods: The maxillae, mandibles, and tibiae of five-week-old male mice were analyzed. Calcification density was analyzed using μCT. The right sides of the specimens were decalcified and processed for Azan staining. The left side of the specimens underwent elemental mapping for Ca, Mg, and P using EPMA.

Results: μCT revealed a significant increase in calcification levels in the following order: enamel, dentin, cortical bone, and trabecular bone. These results reflected the Ca and P levels observed in the EPMA analyses. μCT demonstrated significant differences in the degree of calcification among the enamel tissues or dentin tissues, except for dentin in the maxillary incisors and molars. However, EPMA analysis did not demonstrate significant differences in the Ca and P levels among the same tissue samples.

Conclusions: EPMA elemental analysis can be used to measure Ca and P levels for evaluating the calcification rate of hard tissues. Additionally, the study results validate the evaluation of calcification density via μCT. Furthermore, μCT can evaluate even minute differences in calcification rates compared with EPMA analysis.

Keywords: Biomineralization; Bone; Electron probe microanalysis; Tooth; X-ray computed tomography.