Bone mineral is constituted of biological hydroxyapatite crystals. In developing bone, the mineral crystal matures and the Ca/P ratio increases. However, how an increase in the Ca/P ratio is involved in maturation of the crystal is not known. The relationships among organic components and mineral changes are also unclear. The study was designed to investigate the process of calcification during rat calvarial bone development. Calcification was evaluated by analyzing the atomic distribution and concentration of Ca, P, and C with scanning electron microscopy (SEM)-energy-dispersive X-ray (EDX) spectroscopy and changes in the crystal structure with X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. Histological analysis showed that rat calvarial bone formation started around embryonic day 16. The areas of Ca and P expanded, matching the region of the developing bone matrix, whereas the area of C became localized around bone. X-ray diffraction and FTIR analysis showed that the amorphous-like structure of the minerals at embryonic day 16 gradually transformed into poorly crystalline hydroxyapatite, whereas the proportion of mineral to protein increased until postnatal week 6. FTIR analysis also showed that crystallization of hydroxyapatite started around embryonic day 20, by which time SEM-EDX spectroscopy showed that the Ca/P ratio had increased and the C/Ca and C/P ratios had decreased significantly. The study suggests that the Ca/P molar ratio increases and the proportion of organic components such as proteins of the bone matrix decreases during the early stage of calcification, whereas crystal maturation continues throughout embryonic and postembryonic bone development.

Healing bone is immaturely calcified initially and proceeds calcification gradually, that is, as the bone volume increases, mineral increases in density and matures in quality, while organic components decrease.

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The study was designed to investigate calcification in developing rat mandibular bone using whole mount staining, micro-computed tomography (micro-CT) and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). Wistar rats at embryonic days 16, 18, and 20 and postnatal weeks 1 and 6 were used. Rats were fixed with 4% paraformaldehyde and heads were resected, frozen and sectioned for histology, then analysed with SEM-EDX. Some of the specimens were observed with micro-CT. Other rats were fixed and stained with alcian blue and alizarin red for whole mount staining. Histology and whole mount staining showed that osteoid was deposited around Meckel's cartilage at day 15 and developed into bone at day 16. Accumulation of Ca and P was identified in the bone matrix with SEM-EDX. The area of bone expanded until week 6. The Ca/P ratio increased, whereas the C/Ca and C/P ratios decreased during development. Micro-CT demonstrated an increase in radio-opacity with bone development. The results suggest that rat mandibular bone formation is initiated around Meckel's cartilage at day 15. Deposition and maturation of the calcium phosphate mineral increase gradually with decrease in the organic component as the rat mandible develops.Rat mandibular bone formation begins with deposition of uncalcified bone matrix in the middle of the mandibular process, lateral to Meckel's cartilage, at embryonic day 15 (E15). The uncalcified bone matrix calcifies at E16 (2,3,13,14). Various regions of the mandibular bone differentiate and expand from the ossification site with time. The mandible has been shown to be one of the first bones to calcify in pigs (6, 10) and rabbits (4). Calcification of the bone matrix in the mandible may proceed by deposition, and then by increase and maturation of calcium phosphate minerals, as reported in calvaria (5). However, little information is available about the process of mandibular bone calcification during development. The study was designed to investigate the process of calcification in developing rat mandibular bone. We examined the developing mandible with histology and whole mount staining to demonstrate the spatiotemporal three-dimensional architecture of developing bone. The process of calcification related to mandibular bone development is shown using micro-computed tomography (micro-CT). Analysis of the concentrations and distributions of elements that constitute the bone matrix, that is, calcium (Ca), phosphorus (P) and carbon (C) is performed by scanning electron microscope equipped with an energy dispersive X-ray spectroscope (SEM-EDX).