Fig. 8: Phov-ACP mediates intra- and extra-fibrillar mineralization.

a Cryo-TEM image of a uranyl acetate stained collagen fibril mineralized by Phov-ACP for 6 h. Phov-ACP with an electron-dense nanofibrous structure (asterisk) randomly bound to the collagen. b A stained collagen fibril mineralized for 12 h. ACP (asterisks) infiltrated into the fibril without a discernible preference for band location. Needle-like apatite (arrowhead) could be observed within the intrafibrillar space. c After 48 h of mineralization, the collagen fibril was highly mineralized, with intrafibrillar apatite crystallites (arrowhead) aligned along the fibril’s c-axis and extrafibrillar crystalline (arrow) deposited around the fibril. Scale bars: 100 nm. Experiments were repeated independently (a–c) three times with similar results. d Schematic overview of Phov-mediated hierarchical mineralization. On the mesoscale, Phov-ACP self-assembles into mineral-dense amyloid-like aggregates and spatially binds to the collagen matrix, thereby regulating the mineralized spherule formation and growth. On the nanoscale, Phov-stabilized ACPs adhere to the collagen fibrils. Driven by multiple forces, ACPs are released from Phov and diffuse into the fibril, resulting in collagen intra- and extra-fibrillar mineralization. The precise regulation from the nanoscale cross-fibrillarly mineralized collagen fibrils into the mesoscale mineralized spherules lies in the intricate Phov-mediated hierarchical orchestration. Abbreviation: Phov, phosvitin; ACP, amorphous calcium phosphate; HAP, hydroxyapatite.