The ability to probe fresh tissue is a key feature to biomedical Raman spectroscopy. However, it is unclear how Raman spectra of calcified tissues are affected by freezing. In this study, six transverse sections of femoral cortical bone were subjected to multiple freeze/thaw cycles and probed using a custom Raman microscope. Significant decreases were observed in the amide I and amide III bands starting after two freeze thaw cycles. Raman band intensities arising from proline residues of frozen tissue appeared consistent with fresh tissue after four cycles. Crystallinity values of bone mineral diminished slightly with freezing and were noticeable after only one freezing. Mineral carbonate levels did not deviate significantly with freezing and thawing. The authors recommend freezing and thawing bone tissue only once to maintain accurate results.
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.
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