Bone mineralization and the effects of elevated osteopontin: from symmetry-breaking foci to 3D space-filling tessellation.

Abstract

At the nanoscale, lamellar bone tissue mineralization ensues via heteronucleation of small mineral foci within the osteoid. The foci grow to produce a mature, volume-filling tessellation pattern at the micrometer-scale. Mineralization-inhibiting osteopontin (OPN) mediates this bone mineralization pathway and, eventually, the microscale properties of bone tissue. Using 2D and 3D electron microscopy, here we have assessed how the abundance of OPN can affect nanoscale mineralization, mineral ripening, and microscale patterning of mineral in normal wild-type mouse bone, and we compare that to mutant mouse models having elevated OPN (Fgf23^-/- and Hyp mice). When OPN is elevated, volume-filling mineral tessellation was incomplete (showing a four-fold increase in mineral surface area in the vicinity of the mineralization front in Hyp bone). Immunogold labeling showed excessive OPN in the foci, suggesting an arrest of their growth and an interruption of the pathway towards microscale tessellation. In Fgf23^-/- mice, electron tomography and 3D focused ion beam-scanning electron microscopy (FIB-SEM) imaging of mineral foci show instances of core-shell morphology with crystalline mineral confined to the focus interior, and an amorphous nanogranular texture persisting in the outer shell. Electron energy-loss spectroscopy, which is sensitive to nanoscale elemental composition, showed a lower Ca/P ratio at the periphery of Hyp foci, consistent with a more amorphous mineral character, suggesting that OPN may play a role in delaying the amorphous-to-crystalline transition. These aspects of nanoscale mineral maturation in mutant mice having elevated OPN implicate this protein as a fine-tuning regulator of mineralization kinetics, mineral composition, and mechanical properties of bone.