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Quantitative Comparison of Bone Metabolism Using Fluorescence and X-Ray Microscopy Techniques


Introduction: X-ray computed tomography has been proposed as a non-invasive, high-throughput, 3-D alternative to conventional bone histology, but a direct comparison between these two modalities has not yet been established. In this study, we specifically assess the ability of μCT (micro-computed tomography) to produce metrics of osseous tissue formation using dynamic histomorphometry as a standard for comparison.

Methods: Rat dento-alveolar complexes were implemented as preclinical models to examine formation events in bone, dentin, and cementum. A total of 8 specimens were processed (4 control, 4 ligated), with alternating tetracycline hydrochloride/alizarin red fluorochromes administered over 11 weeks. On week 12, rats were sacrificed, with hemi-maxilla dissected, embedded in Stycast® epoxy, sectioned/ground to ~100μm, and imaged via optical and μCT microscopy. Fluorescence images were collected in 3 channels: brightfield, mCherry, Sapphire, at 20× magnification, while μCT radiographs and tomograms were collected at 90/30 kVp, 20/40×, and single tile/mosaic sizes. Images were taken by tooth–location–tissue, then analyzed via FIJI-ImageJ (line, plot profiles) and Microsoft Excel (linear regressions). Final correlates for dynamic histomorphometry and μCT were mineral apposition rate (MAR) defined as inter–label distance (μm)/time between injections (weeks), and mineral density rate (MDR) defined as change in image gray values (I.a.u.)/distance (μm), respectively.

Results: Cementum had the highest MAR (113 ± 16 μm/week), but lowest MDR (0.03 ± 0.01 I.a.u./μm), while dentin had lowest MAR (34 ± 5 μm/week), but highest MDR (0.09 ± 0.02 I.a.u./μm), with bone being intermediate for both (MAR = 86 ± 20 μm/week, MDR = 0.04 &plus,m; 0.01 I.a.u./μm). MAR vs. MDR revealed the tissue that extended fastest in time simultaneously mineralized the least in space, and vice versa, 1/MAR vs. MDR showed growth and mineralization are positively correlated when normalized to distance, and MAR×MDR calculations produced constant mineral density rates in time (MDRT), suggesting mineralization is a centralized process.

Conclusion: Our study finds μCT and dynamic histomorphometry provide complementary information regarding bone turnover activity, and that tissue apposition dictates mineral distribution, not mineralization rate. Continued research is needed to ascertain whether this relationship holds true for other regions within the skeletal system.

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