Fig. 1.

Nanostructure, composition, and characterization of mineralized collagen. SEM images of (a) non-mineralized and (b) mineralized collagen showing the formation of fibril bundles. Extrafibrillar mineral is apparent in b. (N = 4). Scale bar: 400 nm. Insets: Collagen hydrogels prior to (a) and immediately after (b) a 3-day mineralization period. The mineral formation resulted in a white opaque appearance. c EDX spectra of mineralized samples confirmed the presence of Ca and P in mineralized specimens (bottom), and lack thereof in non-mineralized controls (top). (N = 4). TEM images of (d) non-mineralized collagen showed the typical D-banding pattern of collagen, while the banding becomes obscured in the (e) mineralized collagen, which appears to have both intra and extrafibrillar mineral crystallites (Supplementary Movie 1). Scale bar: 500 nm. (N = 4). f The zoomed view of a single mineralized fibril suggests the preferential orientation of intrafibrillar mineral apatite crystallites (dark streaks) in the (001) position, parallel to the c-axis of the fibril. Scale bar: 50 nm. g Selective area electron diffraction (SAED) of a mineralized collagen shows the typical broad arcs for the (002) plane and overlapping arcs for the (112), (211), and (300) planes that are consistent with the known hexagonal crystalline structure of hydroxyapatite. h FTIR spectra and respective (i) mineral:matrix ratio (****p < 0.0001, Student’s t-test) and (j) crystallinity index (***p < 0.001, Student’s t-test) of mineralized and non-mineralized collagen constructs, dotted lines are reference values of native bone. Data are represented as mean ± SD (N = 6). k AFM nanoindentation modulus of non-mineralized and mineralized hydrated collagen fibrils. Mineralization resulted in over a 1000-fold increase in stiffness for individual fibrils (****p > 0.0001, Student’s t-test) (N = 3). Dotted line is a reference value of native mineralized collagen⁵¹. Source data are provided as a Source Data file