Figure 1.
Pure and mineralized collagen fibrils.
(A) SEM and TEM image of non-crosslinked collagen fibrils showing their native banding patterns. (B) SEM image of non-crosslinked collagen fibrils after mineralization appearing a filamentous substructure. D-banding can be only observed on areas without subfibrillar structure. SEM (C) and AFM (D) image of crosslinked collagen fibrils with native banding patterns. Dotted line in (C) marks the width of a single collagen fibril. The microfibrillar structure is visible with careful observation on the AFM image in (D). (E) (F) and (G) SEM images of crosslinked collagen fibrils after mineralization composed of bundles of subfibrils. (H) A cross-sectional view of crosslinked collagen fibrils after mineralization. Dashed circles in (F) and (H) mark the outer edges of the individual MCFs. Dotted line in G marks the width of a MCF. Some of the multiple subfibrils are pointed by arrows in (E) and (H).
Figure 2.
TEM micrographs of unstained mineralized collagen fibrils.
(A) and (B) typical TEM images of non-crosslinked collagen fibrils after mineralization showing the bundles of subfibrils. (C) Typical TEM image of crosslinked collagen fibrils after mineralization. (D) Dark-Field TEM image of crosslinked collagen fibril after mineralization constructed by selecting one of the (002) arcs with the objective aperture. It illuminates some of the [001] aligned hydroxyapatite single crystals, which appear as short bright strands. (E) Selected area electron diffraction pattern of one mineralized collagen fibril in (C) with labeled lattice planes of hydroxyapatite crystals. (D) A 9.7-nm wide isolated subfibril from crosslinked collagen fibrils after mineralization.
Figure 3.
TEM images of the crosslinked collagen fibrils after mineralization.
(A) A representative TEM micrograph of transversely-sectioned mineralized fibrils that shows hundreds of irregular mineral fragments. (B) Magnified view of subfibrils in the area marked by the dotted square in (A). (C) Lattice image of subfibrils from a crushed MCF showing low mass contrast in the center of the subfibrils. (D) Transversely-sectioned individual subfibril showing low mass contrast in the center. (C) and (D) suggest the formation of a core-shell collagen-HA subfibrillar structure where HA crystals encapsulate collagen microfibrils.
Figure 4.
SEM image of the calcium phosphate mineral.
The mineral was precipitated from a PILP solution after 14 days of incubation without collagen fibrils. Some of the thin crystals with curved shapes are pointed by arrows.
Figure 5.
Comparison of the subfibrillar structure produced in vitro to native bone.
(A) Transverse view of the fractured collagen sheet produced by the PILP process showing hundreds of subfibrils. (B) Fractured surface of bovine cortical bone exhibits similar subfibrils (arrow) with similar size scale to that of the biomimetic mineralized collagen fibrils in (A).
Figure 6.
XRD of MCFs and native hard tissues.
XRD spectra of the crosslinked collagen fibrils after biomimetic mineralization, bovine cortical bone and dentin. Diffraction in all types of samples corresponded to hydroxyapatite nanocrystals. The reconstituted collagen fibrils were compressed as a sheet, resulting in oriented crystals with higher peak intensity for (300) and (210) planes in MCFs than those of native hard tissues.
Figure 7.
TEM micrographs of bovine cortical bone.
(A) and (B) Longitudinally-sectioned bovine bone showing distinct individual hydroxyapatite crystals as dark strands. Multiple long (20-190 nm) crystal strands are paired. The thickness of the crystal strands is 2-4 nm and the separation distance between two strands is approximately 2.5 nm. (C) and (D) Crushed bovine bone showing crystal fragments with widths from 5.7 to 10 nm. Dimensions of the widths of the crystals in (C) and (D) were consistent with those shown in (B).
Figure 8.
Biomimetic mineralized collagen fibrils after heat treatment.
SEM (A) and TEM (B) micrographs of biomimetic mineralized collagen fibrils after heat treatment at 600 °C for 3 hours. Deproteinized fibrils retained their fibrillar structural integrity, although the subfibrils were coalesced within each fibril. A denser packing of crystals with the appearance of periodicity along the longitudinal direction of the fibrils was consistent with the D periodicity of collagen fibrils, which suggests that more mineral was deposited in the gap zones. Arrows point to the dark bands with more mineral deposited. Insert in (B) is the selected area electron diffraction of the fibrils, which indicates that the mineral is still preferentially oriented parallel to the longitudinal axis of the fibril, although the arc has widened (this could be due to the overlap of three fibrils, or the heating process).