Research ArticleMATERIALS SCIENCE

Contribution of biomimetic collagen-ligand interaction to intrafibrillar mineralization

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Science Advances  29 Mar 2019:
Vol. 5, no. 3, eaav9075
DOI: 10.1126/sciadv.aav9075
  • Fig. 1 Characterization of PAA-collagen.

    (A) Infrared spectra of PAA-collagen sponges. Spectra were normalized along the collagen amide A peak (~3300 cm−1, NH stretch coupled with hydrogen bond). Peaks at ~1640, ~1545, and ~1240 cm−1 are assigned to the amide I (C═O stretch), amide II (NH bend coupled with CN stretch), and amide III (NH bend coupled with CN stretch) peaks of collagen, respectively. Peaks at ~2920 cm−1 are assigned to amide B (CH2 asymmetrical stretch) bands. Compared to the spectrum of bare collagen, the spectra of HPAA-bound collagen (HPAA-collagen) and LPAA-bound collagen (LPAA-collagen) sponges show increases in the amide B, I, II, and III peaks. (B) Solid surface ζ potential of HPAA-collagen, LPAA-collagen, and bare collagen. Cross-linking of PAA to the collagen molecule resulted in significantly lower ζ potentials of −17.17 ± 1.98 mV for HPAA-collagen, −15.19 ± 1.22 mV for LPAA-collagen, and −3.04 ± 0.37 mV for bare collagen (P < 0.05 for all pairwise comparisons). (C and D) Comparison of carboxyl (C) and amine groups (D) between PAA-collagen and bare collagen. After anionic modification, the quantities of carboxyl group (in mM/g of collagen) in the HPAA-collagen (1.907 ± 0.084) and LPAA-collagen (1.807 ± 0.035) were significantly higher than that of the bare collagen (0.460 ± 0.017) because of the introduction of additional carboxyl groups to the collagen molecules (P < 0.05). The quantities of amine group in the HPAA-collagen (0.033 ± 0.004) and LPAA-collagen (0.023 ± 0.002) were significantly lower than that of the bare collagen (0.132 ± 0.007) because of the conjugation of free amine groups with PAA via the O═C...N▬H linkage (P < 0.05).

  • Fig. 2 Cryo-EM of mineralization of bare collagen and HPAA-collagen fibrils.

    (A) Grid dipped in freshly prepared HPAA-CaP solution. Scale bar, 50 nm. Prenucleation clusters (open arrowheads) and amorphous calcium phosphate (ACP) droplets (pointers) were randomly distributed within the frozen medium. (B) Grid with bare collagen immersed in HPAA-CaP for 1 hour. Scale bar, 50 nm. Fibrils (open arrows) were not mineralized at this stage. (C to G) HPAA-collagen fibrils were mineralized in unstabilized CaP solution for 1 (C and D), 8 (E), 24 (F), and 72 hours (G). Arrows, prenucleation cluster aggregates; open arrowheads, prenucleation cluster singlets; asterisks, irregular ACP droplets. (C) Circular hole in carbon film (open arrows) shows the concentration of mineral precursors along the periphery of unstained collagen fibrils (arrows). Scale bar, 500 nm. (D) Prenucleation cluster aggregates along the fibril’s surface (arrows) at 1 hour. Scale bar, 100 nm. (E) At 8 hours, fibrils (open arrows) were filled with a slightly more electron-dense material. Scale bar, 50 nm. Prenucleation cluster aggregates were seen in the fibril’s vicinity, while singlets were predominantly located further away. (F) Upper montage of an incompletely mineralized collagen fibril at 24 hours. Scale bar, 100 nm. Bottom left: The intrafibrillar minerals depicted by the pointer [similar to those in (E)] probably represent intrafibrillar ACP that had not yet been transformed into crystalline CaP. Scale bar, 20 nm. Bottom right: Less mineralized part of the fibril with more potentially ACP phases (pointers). Scale bar, 20 nm. (G) Heavily mineralized fibril at 72 hours shows commencement of extrafibrillar mineralization (open arrow). Scale bar, 50 nm. (H to J) 3D rendering of the early (H), middle (I), and late (J) phases of intrafibrillar mineralization of HPAA-collagen showing accumulation of prenucleation cluster aggregates (yellow) along the fibril surface; intrafibrillar minerals are depicted in orange.

  • Fig. 3 Molecular dynamics simulations.

    (A) Profile of bare collagen microfibrillar structures with water molecules within the intrafibrillar spaces. Blue ribbons, collagen triple helices; red dots, water molecules. (B) Side view of bare collagen microfibrils. The blue lines describe the 67-nm-long simulation box; the dimensions of which are a = 24.2 nm, b = 2.83 nm, and c = 67.79 nm with α = 90°, β = 90°, and γ = 105.58°. (C and D) Movement of Ca2+ (yellow spheres), HPO42− (red assemblies), and assembled CaP mineralization precursors (yellow-red assemblies) across the collagen microfibrils with the HPAA (green chain) bound to the LYS1099 amino acids of the collagen molecules. Water molecules, Na+, and Cl ions are not shown for clearer viewing. (E) Top: Simulation of the movement of various ions across the HPAA-collagen microfibrillar structures at designated simulation times. Blue spheres, purple spheres, yellow spheres, red assembly, yellow-red assemblies, and green chain structure represent Na+ ions, Cl ions, Ca2+ ions, HPO42− ions, CaP mineralization precursors, and HPAA, respectively. Bottom: Dynamic changes in the number of Ca2+ (black line), HPO42− (red line), Na+ (green line), and Cl (blue line) in the intrafibrillar and extrafibrillar regions along the a-axis boundary at 0, 30, 50, and 70 ns. (F) Movement of Ca2+, HPO42−, and assembled CaP mineralization precursors across the bare collagen microfibrils with unbound HPAA in the extrafibrillar region (control). (G) Comparison of the distribution of various ions within the intrafibrillar region in the collagen-bound HPAA and the unbound HPAA mineralization models. (H) Comparison of root mean square deviation of collagen molecules in the collagen-bound HPAA and the unbound HPAA models. (I) Comparison of solvent-accessible surface area (SASA) of the collagen molecules between the two models.

  • Fig. 4 AFM of the 3D surface topography and modulus of elasticity (Young’s modulus) mapping of representative air-dried, mineralized bare collagen and HPAA-collagen fibrils.

    (A) Bare collagen fibrils (BC) had a close-to-normal distribution of Young’s modulus transversely across the fibril. A sinusoidal pattern of variation in Young’s modulus was detected lengthwise along the fibril, corresponding to the periodic topographic variations along the fibril’s longitudinal axis. (B) Bare collagen mineralized in CaP solution without nucleation inhibitor (BC + CaP). The transverse Young’s modulus of the CaP group was similar to the BC group. Periodic variation in the lengthwise Young’s modulus was not obvious, which may be attributed to the random deposition of extrafibrillar minerals on the fibril’s surface. (C) Bare collagen mineralized with HPAA-CaP solution (BC + HPAA-CaP). After intrafibrillar mineralization, both the transverse and lengthwise Young’s moduli of the collagen fibril were significantly increased. (D) HPAA-collagen mineralized with CaP solution without nucleation inhibitor (HPAA-Col + CaP). Features were similar to the BC + HPAA-CaP group. (E) Statistical analyses of the Young’s modulus of collagen fibril in the lengthwise and transverse directions (n = 8). For each chart, pairwise comparisons of the differences among the four groups were all significantly different (P < 0.05). Schematic in the background depicts how mapping of a collagen fibril was performed using an AFM tip.

  • Fig. 5 Characterization of mineralized HPAA-collagen sponges and bare collagen sponges.

    (A) TEM of mineralized HPAA-collagen sponges. Left: Dense mineralization in collagen leaflets cross-linked with HPAA and immersed in CaP solution for 7 days (left inset, middle row). Scale bar, 500 nm. Right: Collagen fibrils within leaflet were filled with apatite crystallites oriented along the longitudinal axis of the fibril (between open arrows). Scale bar, 200 nm. (B) TEM of mineralized bare collagen sponges. Left: Heavily mineralized collagen leaflet after immersion in HPAA-CaP solution for 7 days. Nevertheless, the central part of the leaflet was not mineralized (asterisk). Scale bar, 2 μm. Right: Intrafibrillar apatite crystallites arranged along the longitudinal axis of the fibrils. Scale bar, 50 nm. (C) Dynamic attenuated total reflection (ATR)–FTIR shows progressive mineralization of an HPAA-collagen sponge at 12-hour intervals over a 7-day period. Spectra were normalized along the collagen amide I peak (~1640 cm−1). (D) Changes in apatite υ3PO4/collagen amide I ratio of mineralized anionic collagen sponges and unmodified collagen sponges. Compared to bare collagen sponges mineralized in HPAA-CaP solution, HPAA-collagen sponges mineralized in CaP solution demonstrated increases in both the mineralization rate (slope of curves) and extent of mineralization. (E) Dynamic ATR-FTIR shows the progress of mineralization of a bare collagen sponge in HPAA-CaP solution. (F) Stress-strain response of mineralized HPAA-collagen sponges for determining tangent modulus and modulus of toughness (right inset, middle row). (G) Stress-strain response of mineralized bare collagen sponges.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/3/eaav9075/DC1

    Supplementary Materials and Methods

    Fig. S1. Schematic diagram of conjugation of polyanions to bonding of collagen fibrils.

    Fig. S2. Snapshot of the simulation of the Ca2+, HPO42−, and PAA system.

    Fig. S3. Conventional TEM of ruthenium red and uranyl acetate stained collagen fibrils.

    Fig. S4. High performance liquid chromatography of HPAA-collagen in various extraction media.

    Fig. S5. Particle size distribution of the mineralizing solution.

    Fig. S6. Representative TEM images of different mineralization phase of HPAA-collagen.

    Fig. S7. Conventional TEM examination of LPAA-collagen mineralized with CaP.

    Fig. S8. Molecular dynamics simulation of several collagen fibrillar strctures containing bound HPAA molecules.

    Fig. S9. Characterization of mineralized collagen fibrils.

    Fig. S10. Biocompatibility evaluation of bare collagen sponges versus HPAA-collagen sponges.

    Movie S1. Molecular dynamics simulation of aggregation of Ca2+ and HPO42− in the presence of PAA.

    Movie S2. Early phase of intrafibrillar mineralization of HPAA-collagen.

    Movie S3. Middle phase of intrafibrillar mineralization of HPAA-collagen.

    Movie S4. Late phase of intrafibrillar mineralization of HPAA-collagen.

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Schematic diagram of conjugation of polyanions to bonding of collagen fibrils.
    • Fig. S2. Snapshot of the simulation of the Ca2+, HPO42−, and PAA system.
    • Fig. S3. Conventional TEM of ruthenium red and uranyl acetate stained collagen fibrils.
    • Fig. S4. High performance liquid chromatography of HPAA-collagen in various extraction media.
    • Fig. S5. Particle size distribution of the mineralizing solution.
    • Fig. S6. Representative TEM images of different mineralization phase of HPAA-collagen.
    • Fig. S7. Conventional TEM examination of LPAA-collagen mineralized with CaP.
    • Fig. S8. Molecular dynamics simulation of several collagen fibrillar strctures containing bound HPAA molecules.
    • Fig. S9. Characterization of mineralized collagen fibrils.
    • Fig. S10. Biocompatibility evaluation of bare collagen sponges versus HPAA-collagen sponges.
    • Legends for movies S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Molecular dynamics simulation of aggregation of Ca2+ and HPO42− in the presence of PAA.
    • Movie S2 (.mp4 format). Early phase of intrafibrillar mineralization of HPAA-collagen.
    • Movie S3 (.mp4 format). Middle phase of intrafibrillar mineralization of HPAA-collagen.
    • Movie S4 (.mp4 format). Late phase of intrafibrillar mineralization of HPAA-collagen.

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