Fabrication of Amyloid Curli Fibers–Alginate Nanocomposite Hydrogels with Enhanced Stiffness

Abstract

Alginate hydrogels are biocompatible, biodegradable, low-cost, and widely used as bioinks, cell encapsulates, three-dimensional culture matrices, drug delivery systems, and scaffolds for tissue engineering. Nevertheless, their limited stiffness hinders their use for certain biomedical applications. Many research groups have tried to address this problem by reinforcing alginate hydrogels with graphene, carbon nanotubes, or silver nanoparticles. However, these materials present nanotoxicity issues, limiting their use for biomedical applications. Other studies show that electrospinning or wet spinning can be used to fabricate biocompatible, micro- and nanofibers to reinforce hydrogels. As a relatively simple and cheap alternative, in this study we used bioengineered bacteria to fabricate amyloid curli fibers to enhance the stiffness of alginate hydrogels. We have fabricated for the first time bioengineered amyloid curli fibers–hydrogel composites and characterized them by a combination of (i) atomic force microscopy (AFM) to measure the Young’s modulus of the bioengineered amyloid curli fibers and study their topography, (ii) nanoindentation to measure the Young’s modulus of the amyloid curli fibers–alginate nanocomposite hydrogels, and (iii) Fourier-transform infrared spectroscopy (FTIR) to analyze their composition. The fabricated nanocomposites resulted in a highly improved Young’s modulus (up to 4-fold) and showed very similar physical and chemical properties, opening the window for their use in applications where the properties alginate hydrogels are convenient but do not match the stiffness needed.

Engineering Bacillus subtilis for the formation of a durable living biocomposite material

“…Engineering of 'truly' living materials where the living component actively facilitates material fabrication and organization is much more challenging. True ELMs have been created by engineering Escherichia coli to produce an extracellular matrix from curli fibers [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] . Other types of extracellular matrices for ELM fabrication were created from secreted bacterial cellulose to embed microbial cells 26,27 or from elastin-like polypeptides to attach Caulobacter cells via their protein S-layers 28 .…”

Synthetic biology as driver for the biologization of materials sciences

“…Applied to produce hydroxyapatite (HAP)-containing curli-like amyloid fibers [ 131 ] Amyloid curli fibers–alginate nanocomposite Amyloid supplementation to alginate hydrogels for enhanced stiffness. [ 132 ] Electron transfer curli nanofibers Engineered E. coli system for the synthesis of conductive protein networks. [ 133 ] …”

Towards the directed evolution of protein materials

“…As such it is an attractive platform for a variety of applications, such as bioremediation to sequester toxins from the environment 53 , or for in vivo therapeutic use 54 . The mechanical properties of curli fibres have not been extensively studied in isolation; however, they can provide stiffness to natural biofilms 55 , and when added to alginate gels 56 .…”