The Achilles tendon enthesis rebuilds its mineralization front on reloading but retains a nanoscale imprint of unloading

Abstract: The enthesis is a graded fibrocartilaginous interface that transfers load between tendon and bone, yet the nanoscale mechanisms stabilizing its mineralization front remain unclear. Here, we combine multimodal 2D/3D X-ray imaging with nonlinear optical microscopy to map structural, crystalline and extracellular matrix organization across the murine Achilles tendon enthesis under unloading and reloading. Unloading reduces the tidemark-associated two-photon fluorescence (2PF) peak and is accompanied by diffuse mineralization into previously unmineralized fibrocartilage. This unloading-associated mineral exhibits increased apparent crystallite size, an enlarged c-axis lattice parameter, reduced crystalline texture and a diminished collagen order gradient, consistent with an altered mineralization environment. Upon reloading, the 2PF peak recovers, but a new tidemark forms ~20 um from the original boundary, creating a zone with a persistent nanoscale imprint in the mineral tessellation. These findings establish the enthesis as a mechanically governed graded interface in which matrix-mediated boundary control constrains mineral formation and in which a record of mechanical history is imprinted into the nanostructure.