Fig. 2: Characterization of the mineralized spherules induced by Phov-ACP.

a Scheme of Phov-ACP preparation and collagen mineralization. Created in BioRender. Zhang, Y. (2024) https://BioRender.com/s17v456. b Cryo-TEM and SAED images of freshly prepared Phov-ACP complex. High-magnification image (ii) of the area indicated by the red rectangle in (i) showing discernible electron-dense granules (red circle). The SAED (iii) pattern of the area indicated by the red circle in (ii) validated the amorphous state of Phov-ACP. Scale bars: i, ii 50 nm; iii 2 1/nm. c Cryo-TEM and SAED images of a single collagen fibril that had been immersed in Phov-ACP for 24 h. High-magnification image (ii) showing a dense arrangement of needle-shaped mineral crystallites within the fibril. The SAED (iii) pattern of the area indicated by the red circle in (ii) showed (002) diffraction plane, indicating oriented apatite crystallites were aligned with the longitudinal axis of the fibril. Scale bars: i, ii 200 nm; iii 2 1/nm. d FIB-SEM images of the 3D collagen membrane after 48 h of mineralization by Phov-ACP. Mineralized spherules were evenly distributed within the collagen matrix. High-magnification images showed arrays of intrafibrillarly mineralized collagen fibrils constructed the mineralized spherules. Scale bars: i, ii 5 μm; iii 1 μm. Experiments were repeated independently (b–d) three times with similar results. e EDS elemental mapping of the area framed in (d–ii) showing the spatial distribution of calcium, phosphorus, and oxygen in the spherical inorganic-organic hybrid entity. Scale bar: 5 μm. f–i AFM measurement of nonmineralized 3D collagen membrane and samples that had been mineralized for 48 h and 72 h. 3D surface topography (f) combined with height images (g, h) of the spherules showed an increase in height. Stiffness maps (i) showed that the stiffness increased at mineral deposition sites as mineralization proceeded.