Self-assembly and mineralization of genetically modifiable biological nanofibers driven by β-structure formation

Abstract

Bioinspired mineralization is an innovative approach to the fabrication of bone biomaterials mimicking the natural bone. Bone mineral hydroxylapatite (HAP) is preferentially oriented with c-axis parallel to collagen fibers in natural bone. However, such orientation control is not easy to achieve in artificial bone biomaterials. To overcome the lack of such orientation control, we fabricated a phage-HAP composite by genetically engineering M13 phage, a nontoxic bionanofiber, with two HAP-nucleating peptides derived from one of the noncollagenous proteins, Dentin Matrix Protein-1 (DMP1). The phage is a biological nanofiber that can be mass produced by infecting bacteria and is nontoxic to human beings. The resultant HAP-nucleating phages are able to self-assemble into bundles by forming β-structure between the peptides displayed on their side walls. The β-structure further promotes the oriented nucleation and growth of HAP crystals within the nanofibrous phage bundles with their c-axis preferentially parallel to the bundles. We proposed that the preferred orientation resulted from the stereochemical matching between the negatively charged amino acid residues within the β-structure and the positively charged calcium ions on the (001) plane of HAP crystals. The self-assembly and mineralization driven by the β-structure formation represent a new route for fabricating mineralized fibers that can serve as building blocks in forming bone repair biomaterials and mimic the basic structure of natural bones.

Figure 1

TEM images and circular dichroism spectra of engineered phages. (a) E phages; (b) Q phage bundles; (c) EQ phage bundles. (d) Circular dichroism spectra of wild type, E, Q and EQ phages.

Figure 2

TEM images of nucleation of HAP on E and Q phages after incubation in supersaturated HAP solution for 10 days and 20 days. (a) HAP nucleation on E phages for 10 days. (b) HAP nucleation on E phages for 20 days. (c) HAP nucleation on Q phages for 10 days. (d) HAP nucleation on Q phages for 20 days.

Figure 3

TEM images of the HAP nucleation on EQ phage bundles after incubation in supersaturated HAP solutions for 5 days (a), 10 days (b), 15 days (c) and 20 days (d).

Figure 4

The proposed three dimensional models of possible HAP nucleation mechanism. (a) 3D model of possible EQ phage bundles. (b) The simulation of intermolecular β-sheet formed by two HAP nucleating peptides displayed on the major coat of phage. This model includes the aspartic acid (D) which is the amino acid right following the inserted foreign peptides in pVIII due to its possible contribution to the β-sheet formation. (c) The possible mechanism of the HAP nucleation and growth along c-axis due to the specific match between the negatively charged amino acid in HAP nucleating peptides forming intermolecular β-sheet and the (001) plane of HAP crystals. (d) Schematic illustration showing that the four calcium ions on HAP (001) plane can perfectly match four negatively charged amino acids. (e) A closer look of electrostatic attraction between matched negatively charged amino acids and calcium ions. (f) The 3D model schematically illustrating the possible mechanism of oriented nucleation and growth of HAP crystals triggered by EQ phage bundles. (All the figures were made in PyMOL)

Scheme 1

Phage display technique. (a) TEM image of wild type M13 phages. (b) Display of a foreign peptide on the side wall of M13 phage by inserting the peptide into the N-terminus of major coat protein (pVIII) of M13 phage. (c) 3D model of pVIII (PDB ID: 1ifd).