Mineralization of Developing Mouse Calvaria as Revealed by Raman Microspectroscopy

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Abstract
Raman microspectroscopy is a nondestructive vibrational spectroscopic technique that permits the study of organic and mineral species at micron resolution, offers the ability to work with hydrated and dehydrated specimens in vivo or in vitro, and requires minimal specimen preparation. We used Raman microspectroscopy to determine the composition of the mineral environments present in mouse calvaria, the flat bones that comprise the top of the skull. We have acquired Raman transects (lines of point spectra) from mouse calvaria during a developmental time course ranging from embryonic day 13.5 (E13.5; 6 days before birth) to 6 months of age. Exploratory factor analysis (FA) reveals the presence of a variety of apatitic mineral environments throughout the tissue series. The earliest mineral is observed in the fetal day 15.5 (F15.5) mice and is identified as a carbonated apatite. The presence of a heterogeneous mineralized tissue in the postnatal specimens suggests that ionic incorporation and crystal perfection in the lattice vary as the mouse develops. This variation is indicative of the presence of both recently deposited mineral and more matured remodeled mineral. Band area ratios reveal that the mineral/matrix ratio initially increases, reaches a plateau, and then increases again. The carbonate/phosphate band area ratio remains constant from F18.5 to postnatal day 3 (PN3) and then increases with age. Insights into the chemical species, the degree of mineralization, and the multiple mineral environments that are present in normal calvarial tissue will enable us to better understand both normal and abnormal mineralization processes.