Calcification in rat developing mandibular bone demonstrated by whole mount staining, micro-computed tomography and scanning electron microscopy with energy dispersive X-ray spectroscopy

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.