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Raman spectroscopic evidence of crystalline phosphate precursor to bone apatitic mineral

Bone is a highly specialized connective tissue comprised of cross-linked collagen fibers interspersed with apatitic mineral crystallites of various sizes, shapes, orientation, and composition. However, the nucleation, growth, and propagation of mineral crystallite into the collagenous matrix are not clearly understood. By using a research grade inverted microscope fitted with a line-shaped 830 nm laser and spectrograph, we show that the Raman scatter from mineralizing cell cultures in an incubation chamber can be collected and monitored directly through the bottom of the well-plates over a period of 24 hours. In our studies, murine-derived MC3T3 cells were incubated at 37°C in the presence of 5% CO2 and 85% humidity. Results show a gradual shift in the phosphate ν1 apatitic band center (955-957 cm-1) during the first hour of mineralization. The phosphate ν1 apatitic band width also narrowed during this time. To quantify the amount of crystal growth in vivo, we used a calibration curve derived from X-ray powder diffraction and Raman studies performed on a series of synthetic carbonated apatites and deproteinated mouse femoral specimens. Mineralization in neonatal mouse calvarial culture was observed along the lambdoid suture. Deposition proceeded in a stepwise fashion over the course of ~30 h.