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.

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 (circadian time). We also established the kinetics of mineral deposition in developing bone by using non-invasive 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 6 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.

Abstract. 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. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

Osteogenesis imperfecta (OI) is a heritable bone dysplasia characterized by increased skeletal fragility. Patients are often treated with bisphosphonates to attempt to reduce fracture risk. However, bisphosphonates reside in the skeleton for many years and long-term administration may impact bone material quality. Acutely, there is concern about risk of non-union of fractures that occur near the time of bisphosphonate administration. This study investigated the effect of alendronate, a potent aminobisphosphonate, on fracture healing. Using the Brtl/+ murine model of type IV OI, tibial fractures were generated in 8-week-old mice that were untreated, treated with alendronate before fracture, or treated before and after fracture. After 2, 3, or 5 weeks of healing, tibiae were assessed using microcomputed tomography (μCT), torsion testing, quantitative histomorphometry, and Raman microspectroscopy. There were no morphologic, biomechanical or histomorphometric differences in callus between untreated mice and mice that received alendronate before fracture. Alendronate treatment before fracture did not cause a significant increase in cartilage retention in fracture callus. Both Brtl/+ and WT mice that received alendronate before and after fracture had increases in the callus volume, bone volume fraction and torque at failure after 5 weeks of healing. Raman microspectroscopy results did not show any effects of alendronate in wild-type mice, but calluses from Brtl/+ mice treated with alendronate during healing had a decreased mineral-to-matrix ratio, decreased crystallinity and an increased carbonate-to-phosphate ratio. Treatment with alendronate altered the dynamics of healing by preventing callus volume decreases later in the healing process. Fracture healing in Brtl/+ untreated animals was not significantly different from animals in which alendronate was halted at the time of fracture.

Mechanical loading is integral to the repair of bone damage. Osteocytes are mechanosensors in bone and participate in signaling through gap junction channels, which are primarily comprised of connexin 43 (Cx43). Nitric oxide (NO) and prostaglandin E2 (PGE2) have anabolic and catabolic effects on bone, and the secretion of these molecules occurs after mechanical stimulation. The effect of age on the repair of bone tissue after damage and on the ability of regenerated bone to transduce mechanical stimulation into a cellular response is unexplored. The goal of this study was to examine (1) osteocytes and their mineralized matrix within regenerated bone from aged and mature animals and (2) the ability of regenerated bone explants from aged and mature animals to transduce cyclic mechanical loading into a cellular response through NO and PGE2 secretion. Bilateral cortical defects were created in the diaphysis of aged (21-month-old) or mature (6-month-old) male rats, and new bone tissue was allowed to grow into a custom implant of controlled geometry. Mineralization and mineral-to-matrix ratio were significantly higher in regenerated bone from aged animals, while lacunar and osteocyte density and phosphorylated (pCx43) and total Cx43 protein were significantly lower, relative to mature animals. Regenerated bone from mature rats had increased pCx43 protein and PGE2 secretion with loading and greater NO secretion relative to aged animals. Reduced osteocyte density and Cx43 in regenerated bone in aged animals could limit the establishment of gap junctions as well as NO and PGE2 secretion after loading, thereby altering bone formation and resorption in vivo.