Dynamics of Mineralization during Bone Development.

Abstract

Mineralization is an important process in bone growth, development and regeneration. Mineral deposition and crystallization in tissue are protein-mediated processes where the properties of the mineral are largely dictated by the function of specific proteins. Currently, the mechanism of mineralization is a hotly debated topic. The focus of this thesis is to better understand the dynamic process of mineral formation in bone and to identify physiological influences that affect the mineral pathway or the mineral product. First, the effect of carbonate substitution on crystal properties was explored using a series of B-type carbonated apatites (0.2–10.5 wt% CO32-). It was determined that apatite crystal size is dependent on carbonate substitution due to the amount of strain it puts on the crystal lattice. Next, mineralization kinetics was observed in actively mineralizing ex vivo neonatal murine calvaria using Raman spectroscopy as a real-time monitor of mineral properties. Periodic deposition of apatitic mineral occurred with near-daily frequency for up to 6 days. Timing of the mineral events were compared to the expression of circadian-clock component Period1 using a firefly luciferase bioluminescence reporter gene. The calvaria most actively deposited mineral in the early evening around 9pm. These experiments were repeated in calvaria infected with an adenovirus carrying the bone sialoprotein (BSP) gene. The resulting overexpression of BSP significantly modified the mineralization pathway. BSP induced formation of amorphous calcium phosphate during the first 30 hours followed by a transformation into small hydrated crystals over 40 hours duration. Mineral growth was also observed in fractured murine tibiae after 5 weeks of healing. Both wild-type and Brtl/- mice were screened for mineral crystallinity and carbonate content. Brtl/- mice experienced a delay in mineralization by containing underdeveloped mineral with poor crystallinity and high carbonate content. Lastly, fresh murine femora were frozen and thawed several times to test the consistency of Raman bone quality metrics through the preservation of unfixed bone tissue. Collagen-matrix metrics were significantly affected after four freeze/thaw cycles. Mineral metrics were also affected but to a lesser degree. For Raman studies of fresh bone, one freeze/thawing is recommended.