RESUMO
Solid-state (magic-angle spinning) NMR spectroscopy is a useful tool for obtaining structural information on bone organic and mineral components and synthetic model minerals at the atomic-level. Raman and 31P NMR spectral parameters were investigated in a series of synthetic B-type carbonated apatites (CAps). Inverse 31P NMR linewidth and inverse Raman PO43- ν1 bandwidth were both correlated with powder XRD c-axis crystallinity over the 0.3-10.3 wt% CO32- range investigated. Comparison with bone powder crystallinities showed agreement with values predicted by NMR and Raman calibration curves. Carbonate content was divided into two domains by the 31P NMR chemical shift frequency and the Raman phosphate ν1 band position. These parameters remain stable except for an abrupt transition at 6.5 wt% carbonate, a composition which corresponds to an average of one carbonate per unit cell. This near-binary distribution of spectroscopic properties was also found in AFM-measured particle sizes and Ca/P molar ratios by elemental analysis. We propose that this transition differentiates between two charge-balancing ion-loss mechanisms as measured by Ca/P ratios. These results define a criterion for spectroscopic characterization of B-type carbonate substitution in apatitic minerals.
RESUMO
Osteoblasts, which orchestrate the deposition of small apatite crystals through the expression of nucleating proteins, have been shown to also express clock genes associated with the circadian signaling pathway. We hypothesized that protein-mediated bone mineralization may be linked to circadian oscillator mechanisms functioning in peripheral bone tissue. In this study, Per1 expression in ex vivo neonatal murine calvaria organ cultures was monitored for 6 days using a Per1-luciferase transgene as a bioluminescent indicator of clock function. Fluctuations in Per1 expression had a period of 25 ± 4 hours (n = 14) with early expression at CT09:59 ± 03:37 (CT = circadian time). We also established the kinetics of mineral deposition in developing bone by using noninvasive Raman microscopy to track mineral accumulation in calvarial tissue. The content and quality of newly deposited mineral was continually examined at the interparietal bone/fontanel boundary for a period of 6 days with 1-hour temporal resolution. Using this approach, mineralization over time exhibited bursts of mineral deposition followed by little or no deposition, which was recurrent with a periodicity of 26.8 ± 9.6 hours. As many as six near-daily mineralization events were observed in the calvaria before deposition ceased. Earliest mineralization events occurred at CT16:51 ± 03:45, which is 6 hours behind Per1 expression. These findings are consistent with the hypothesis that mineralization in developing bone tissue is regulated by a local circadian oscillator mechanism.
Assuntos
Calcificação Fisiológica/fisiologia , Ritmo Circadiano/fisiologia , Crânio/fisiologia , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Calcificação Fisiológica/genética , Ritmo Circadiano/genética , Cristalização , Regulação da Expressão Gênica , Camundongos , Técnicas de Cultura de Órgãos , Análise Espectral RamanRESUMO
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.