Genetically Engineered Nanofiber-Like Viruses For Tissue Regenerating Materials

Abstract

Controlling structural organization and signaling motif display of biomimetic matrices at the nanometer scale is of great importance to the functional design of tissue regenerating materials. We have genetically engineered M13 bacteriophage (phage), naturally occurring nanofiberlike viruses, to display a high density of cell-signaling peptides on their major coat proteins. Structural orientation of these phage building blocks can be achieved due to their long-rod shape and monodispersity, which lead them to self-assemble into directionally organized liquid crystalline-like materials. We showed that the constructed viral nanofiber scaffolds were able to support neural progenitor cell proliferation and differentiation as well as direct orientation of their growth in three dimensions. Such functionalized and structurally aligned phage matrices offer promising opportunities for therapies that address challenging medical problems, such as nerve tissue regeneration after spinal cord injuries, or as in vitro model systems for studying complicated cell signaling environments.Designing biomimetic matrices with a precise control of structural organization and display of signaling motifs to regulate cell behavior is one of the most critical issues for the development of tissue regenerating materials. In nature, a nanofibrous protein network, in combination with neighboring cells, support and guide cellular behavior through a multitude of chemical and physical cues. [1][2][3][4] Similarly, manmade materials are being developed to recreate an environment that would provide for comparable controls over cell activities. [5][6][7][8][9][10][11] Recently developed top-down and bottomup material synthesis approaches 6,[8][9][10][11][12][13][14][15] are allowing for biomimetic materials to approximate a level of complexity observed in the natural extracellular environment. Nanofibers offer increased surface area for cell engagement, 10,13 which has led to improved cell attachment and material integration, as well as enhanced cell proliferation and differentiation. 6,12,13 Controlling the density of displayed signaling molecules at the nanometer scale has permitted quantitative investigations of cell behaviors. 9,11,12,[16][17][18] Observations of cell growth on substrates with aligned topographical features, such as nanofibers or ridges, have verified that cells can sense and respond to ordered nanoscale textures with polarization and coalignment. 6,13,19 Furthermore, self-assembling peptide-based materials have been utilized for convenient cell entrapment in three dimensions and offer attractive opportunities for minimally invasive therapy applications. 12,17 These various engineering approaches provide different combinations of advantageous material attributes, yet no single technique has been able to simultaneously satisfy the following multiple requirements of tissue regenerating scaffolds: to provide a cell conducive environment, to display signaling molecules in a controlled manner, to form self-organized nanofilamen...

Biomimetic virus-based colourimetric sensors

“…Phages generate identical copies of themselves through infection of bacterial host cells. Because of its well-defined, monodisperse shape and its ability to display functional peptides, M13 phage has been utilized to fabricate various functional nanomaterials for semiconductor [26][27][28] , energy [29][30][31] and bioengineering applications 32 . Recently, inspired by self-templated materials assembly processes in nature, we developed a process to fabricate an array of hierarchical phage-based structures through controlled extraction d 2 ) and coherent scattering.…”

“…To incorporate the TNT-binding peptide, we genetically engineered the M13 phage's major coat proteins (pVIII). The desired peptide sequences were inserted between the first and the sixth amino acids of the amino terminus of wild-type pVIII, replacing residues 2-5 (Ala-GluGly-Asp-Asp-Pro to Ala-(Insert)-Pro) 32 . To incorporate the most stable phage to carry the consensus TNT-binding peptide (WHWQ) identified by phage display, we designed a partial library with sequence of the form AXXWHWQXXDP using the primer: 5 0 -ATATATCTGCAGNKNNKTGGCATTGGCAGNNKN NKGATCC CGCAAAAGCG GCCTTTAACTCCC-3 0 and the primer 5 0 -GCTGTCTTTCGC TGC AGAGGGTG-3 0 to linearize the vector (N ¼ A/C/G/T and K ¼ G/T).…”

Bioinspired supramolecular fibers drawn from a multiphase self-assembled hydrogel

“…Supramolecular pathways enable the formation of filamentous soft materials that are showing promise in biomedical applications (4)(5)(6), such as cell culture (7)(8)(9) and tissue engineering (10). However, such materials are constrained by the length scale (submicrometer level) (11)(12)(13), energy intake during production (9), and complex design of assembly units (14).…”

Calcifying tissue regeneration via biomimetic materials chemistry

“…Bacteriophages have been genetically engineered to present peptides on their outer casings (capsid) that promulgate crystal binding [49,64]. Decoration of capsids with cell signalling or adhesion peptides offers a chance to create ordered structures that may order cell adhesion density and cell distribution [65]. Intricately patterned chains of nanocrystals have been organized onto naturally fabricated protein films derived from bacterial cell envelopes (surface-layers or S-layers) in cadmium sulfide [66].…”

“…Inorganic nanocoatings have also been implemented with human primary mesenchymal stem cells facilitating induction towards specialization into a bone cell lineage and targeted gene transfer into human MSCs [65]. Cell growth allowed to proceed on the surface textures shows them to proliferate and spread in the alignment with the underlying fibres.…”