During crucial growth stages of vertebrate long bones, calcified cartilage beneath the growth plate is anchored to bone by a third mineralized component, the cement line. Proper skeletal development is contingent on the interplay of these three constituents, yet their mineralization processes and structural interactions are incompletely understood, in part from limited knowledge of their meso‐ and nanoscale features. Herein, focused ion beam‐scanning electron microscopy (FIB‐SEM) with serial surface imaging is applied to examine the cartilage–bone interface of mouse femoral heads at an unprecedented scale: FIB‐SEM provides 3D, nanometer resolution of structural details for volumes encompassing metaphyseal calcified cartilage, bone, and the intervening cement line. A novel and complex structural network is revealed, comprising densely packed nanochannels smaller than bone canaliculi (≈10–50 nm diameter) within the calcified cartilage and bone extracellular matrices, but absent in the cement line. A structural correlation is demonstrated between the nanochannels and ellipsoidal mineral domains, which appear to coalesce during mineralization in a process analogous to powder sintering in metallurgy. A mineralization process is proposed, supported by energy‐dispersive X‐Ray spectroscopy of nanochannel contents, in which these unreported structures offer ion and molecule conduits to access the extracellular matrices of calcified cartilage and bone.

During skeletal development, bone growth and mineralization require transport of substantial amounts of calcium, while maintaining very low concentration. How an organism overcomes this major logistical challenge remains mostly unexplained. To shed some light on the dynamics of this process, cryogenic focused ion beam‐scanning electron microscopy (cryo‐FIB/SEM) is used to image forming bone tissue at day 13 of a chick embryo femur. Both cells and matrix in 3D are visualized and observed as calcium‐rich intracellular vesicular structures. Counting the number of these vesicles per unit volume and measuring their calcium content based on the electron back‐scattering signal, the intracellular velocity at which these vesicles need to travel to transport all the calcium required for the mineral deposited in one day within the collagenous tissue can be estimated. This velocity at 0.27 µm s⁻¹ is estimated, which is too large for a diffusion process and rather suggests active transport through the cellular network. It is concluded that calcium logistics is hierarchical and based on several transport mechanisms: first through the vasculature using calcium‐binding proteins and the blood flow, then active transport over tens of micrometers through the network of osteoblasts and osteocytes, and finally diffusive transport over the last one or two microns.