Transformation of bone mineral morphology: From discrete marquise-shaped motifs to a continuous interwoven mesh

Continual bone apposition at the cranial sutures provides the unique opportunity to understand how bone is built. Bone harvested from 16-week-old Sprague Dawley rat calvaria was either (i) deproteinised to isolate the inorganic phase (i.e., bone mineral) for secondary electron scanning electron microscopy or (ii) resin embedded for X-ray micro-computed tomography, backscattered electron scanning electron microscopy, and micro-Raman spectroscopy. Interdigitated finger-like projections form the interface between frontal and parietal bones. Viewed from the surface, bone mineral at the mineralisation front is comprised of nanoscale mineral platelets arranged into discrete, ~0.6-3.5 μm high and ~0.2-1.5 μm wide, marquise-shaped motifs that gradually evolve into a continuous interwoven mesh of mineralised bundles. Marquise-shaped motifs also contribute to the burial of osteoblastic-osteocytes by contributing to the roof over the lacunae. In cross-section, apices of the finger-like projections resemble islands of mineralised tissue, where new bone apposition at the surface is evident as low mineral density areas, while the marquise-shaped motifs appear as near-equiaxed assemblies of mineral platelets. Carbonated apatite content is higher towards the internal surface of the cranial vault. Up to 4 μm from the bone surface, strong Amide III, Pro, Hyp, and Phe signals, distinct PO₄ ^3- bands, but negligible CO₃ ^2- signal indicate recent bone formation and/or delayed maturation of the mineral. We show, for the first time, that the extracellular matrix of bone is assembled into micrometre-sized units, revealing a superstructure above the mineralised collagen fibril level, which has significant implications for function and mechanical competence of bone.