Protein disorder-order interplay to guide the growth of hierarchical mineralized structures - PubMed (original) (raw)

doi: 10.1038/s41467-018-04319-0.

Sherif Elsharkawy 1 2 3, Maria F Pantano 5, Esther Tejeda-Montes 2, Khushbu Mehta 2, Hasan Jamal 2, Shweta Agarwal 6 7 8, Kseniya Shuturminska 1 3, Alistair Rice 6, Nadezda V Tarakina 2, Rory M Wilson 2 4, Andy J Bushby 2 4, Matilde Alonso 9, Jose C Rodriguez-Cabello 9, Ettore Barbieri 2 10, Armando Del Río Hernández 6, Molly M Stevens 6 7 8, Nicola M Pugno 2 5 11, Paul Anderson 1 3, Alvaro Mata 12 13

Affiliations

Protein disorder-order interplay to guide the growth of hierarchical mineralized structures

Sherif Elsharkawy et al. Nat Commun. 2018.

Abstract

A major goal in materials science is to develop bioinspired functional materials based on the precise control of molecular building blocks across length scales. Here we report a protein-mediated mineralization process that takes advantage of disorder-order interplay using elastin-like recombinamers to program organic-inorganic interactions into hierarchically ordered mineralized structures. The materials comprise elongated apatite nanocrystals that are aligned and organized into microscopic prisms, which grow together into spherulite-like structures hundreds of micrometers in diameter that come together to fill macroscopic areas. The structures can be grown over large uneven surfaces and native tissues as acid-resistant membranes or coatings with tuneable hierarchy, stiffness, and hardness. Our study represents a potential strategy for complex materials design that may open opportunities for hard tissue repair and provide insights into the role of molecular disorder in human physiology and pathology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1

Fig. 1

ELR spherulites and hierarchical mineralized structures. a Photograph of a transparent, robust, and flexible statherin-ELR membrane before mineralization. The membrane’s cross-section before mineralization b imaged by SEM and c histologically stained displaying a positive staining for both β-amyloid fibers and d elastin fibers with Congo Red and Elastin von Gieson stains, respectively. e SEM image of the membrane’s cross-section comprising of ELR nanofibers. f, g Polarized light microscope (PLM) images depicting the presence of ELR spherulites with the characteristic birefringent Maltese-cross pattern on the surface and within the bulk of the membranes. h-k SEM images of the top of an RGDS-ELR membrane after mineralization showing the hierarchical organization of the mineralized structures including h aligned fluorapatite nanocrystals that are i, j grouped into prism-like microstructures that further grow into k macroscopic circular structures. l, m The hierarchical structures grow until they meet each other, and n can mineralize completely thin membranes. o Rietveld refinement of an XRD pattern of mineralized membranes showing the fluorapatite nature of the crystalline phase with the typical Bragg peaks of apatite. p 19F solid-sate MAS-NMR spectra confirming the presence of fluorapatite and CaF2 (fluorite) phase at – 103 and – 108 p.p.m., respectively, with increasing fluorapatite peak intensity on the mineralized membrane (green) compared with those without the ELP membrane (red) at the same conditions. q Young’s modulus and hardness relationship between the mineralized structures and different mineralized tissues. Scale bars: a 5 mm; b 20 µm; c, d 40 µm; e 200 nm; f 10 µm; g 3 µm; h 200 nm; i 1 µm; j 10 µm; k 20 µm; l 30 µm; l (inset) 20 µm; m 5 µm; n 20 µm

Fig. 2

Fig. 2

Bulk characterization. Organic–inorganic interactions within the bulk of the mineralized membranes. SEM observations revealed a the abundant presence of a dense pattern of spherulite-like structures with b a granulated central region at the bulk of the membranes’ cross-sections, which c template the growth of fluorapatite spherulites. d Near the membrane surface, mineralized structures with nanocrystals grow vertically toward the surface of the membrane. DDC-SEM images of e the core of fluorapatite spherulite structures located closer to the surface of the membrane and f deeper within the bulk of the membrane, revealing a thin low-density material (green) surrounding a denser (orange) one. g Backscattered electron (BSE) image showing brighter areas at the center of the structures, indicating the presence of mineral deep within the membrane. h, i FIB sectioning of the hierarchically mineralized structure resolving the deeper mineralized core structures located underneath the center of the structures. j, kTEM images from a FIB milling lift-out of a mineralized structure illustrating j the change in growth direction of the nanocrystals from parallel to the surface towards the bulk of the ELR membrane, and k the growing interface between the flat-ended inorganic crystals and the organic ELR material. l HRTEM image of a single fluorapatite crystal showing its growth orientation and crystal lattices. m SAED and n FFT analyses showing the characteristic diffraction pattern of the fluorapatite crystals (left) and its 10° co-alignment, respectively. Scale bars: a 2 µm; b, c 1 µm; d 2 µm; e, f 200 nm; g 20 µm; h, i 5 µm; j 200 nm; k 100 nm; l 10 nm; m 3 nm−1

Fig. 3

Fig. 3

Protein disorder–order optimization and process tuneability. a, b Graphs showing the different levels of ELR order and disorder as a function of cross-linking. The levels of ELR ordered β-sheet structure and disordered random coil can be modulated and tuned, while maintaining β-turn and α-helix conformations nearly constant. c Table summarizing the tuneability of the process including during both ELR matrix assembly (Stage I) and the CaP nucleation and growth (Stage II). Scale bars: 3 µm (ELR spherulite morphology), 20 µm (apatite hierarchical structures), and 200 nm (nanocrystal density)

Fig. 4

Fig. 4

Mechanism of formation. A schematic illustrating the two stages of the proposed mechanism divided in (I) ELR matrix assembly and (II) calcium phosphate (CaP) nucleation and growth

Fig. 5

Fig. 5

Resemblance between enamel and the hierarchical structures. SEM images illustrating the close resemblance of human dental enamel a to the hierarchically ordered mineralized structures b grown on a RGDS-ELR membrane at multiple length scales. Scale bars: a, b 200 nm; c, d 1 µm; and e, f 20 µm

Fig. 6

Fig. 6

Dental applications of the hierarchical structures. a Application of the in-situ cross-linked ELR membrane conformed over the rough and uneven surface of exposed human dentin, exhibiting the hierarchical mineralized structures as a coating on top of the native tissue. SEM images depicting b a removed section (white square) of the mineralized membrane with c the nanocrystals infiltrating, binding, and occluding the open dentinal tubule structures. d FIB milling of the mineralized coating at different depths to observe the dentin-membrane interface, which exhibits infiltration of nanocrystals occluding the dentinal tubules. e SEM images revealing the effect of the acid attack at different time-points (15 min, and 7 days) on both human dental enamel and the hierarchical mineralized structures. f Graph illustrating the Young’s modulus and hardness of the human enamel and mineralized structures after the acid attack. g DDC-SEM images of the hierarchical mineralized structures after the enzymatic digestion, showing a partially remaining organic material (arrow). Scale bars: a, b 50 µm; c 3 µm; d 10 µm; e 1 µm; g 500 nm. Error bars are represented as SD, n = 10

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