Scaffolds Formed via the Non-Equilibrium Supramolecular Assembly of the Synergistic ECM Peptides RGD and PHSRN Demonstrate Improved Cell Attachment in 3D

Abstract

Self-assembling peptides (SAPs) are a relatively new class of low molecular weight gelators which immobilize their solvent through the spontaneous formation of (fibrillar) nanoarchitectures. As peptides are derived from proteins, these hydrogels are ideal for use as biocompatible scaffolds for regenerative medicine. Importantly, due to the propensity of peptide sequences to act as signals in nature, they are easily functionalized to be cell instructive via the inclusion of bioactive epitopes. In nature, the fibronectin peptide sequence, arginine-glycine-aspartic acid (RGD) synergistically promotes the integrin α 5 β 1 mediated cell adhesion with another epitope, proline-histidine-serine-arginine-asparagine (PHSRN); however most functionalization strategies focus on RGD alone. Here, for the first time, we discuss the biomimetic inclusion of both these sequences within a self-assembled minimalistic peptide hydrogel. Here, based on our work with Fmoc-FRGDF ( N -flourenylmethyloxycarbonyl phenylalanine-arginine-glycine-aspartic acid-phenylalanine), we show it is possible to present two epitopes simultaneously via the assembly of the epitopes by the coassembly of two SAPs, and compare this to the effectiveness of the signals in a single peptide; Fmoc-FRGDF: Fmoc-PHSRN ( N -flourenylmethyloxycarbonyl-proline-histidine-serine-arginine-asparagine) and Fmoc-FRGDFPHSRN ( N -flourenylmethyloxycarbonyl-phenylalanine-arginine-glycine-asparticacid-phenylalanine-proline-histidine-serine-arginine-asparagine). We show both produced self-supporting hydrogel underpinned by entangled nanofibrils, however, the stiffness of coassembled hydrogel was over two orders of magnitude higher than either Fmoc-FRGDF or Fmoc-FRGDFPHSRN alone. In-vitro three-dimensional cell culture of human mammary fibroblasts on the hydrogel mixed peptide showed dramatically improved adhesion, spreading and proliferation over Fmoc-FRGDF. However, the long peptide did not provide effective cell attachment. The results demonstrated the selective synergy effect of PHSRN with RGD is an effective way to augment the robustness and functionality of self-assembled bioscaffolds.

Engineered mosaic protein polymers; a simple route to multifunctional biomaterials

“…Since the Caf1 subunit is only 6 nm long a 10 μm diameter cell will interact with hundreds or thousands of Caf1 subunits so even if they are present at 1% of a mosaic polymer, poorly expressing versions are likely to be biologically active. Finally, some motifs exhibit synergy when in close proximity to each other, for example the RGDS and PHSRN motifs of fibronectin [38, 39]. Due to the 6 nm subunit repeat, the production of mosaic polymers will result in the random close proximity of Caf1 subunits harbouring different motifs in a way that a mixture of single subunit polymers might not.…”

Enhancing Peptide Biomaterials for Biofabrication

“…The ability to manipulate the physical and mechanical properties of synthetic SAP hydrogels by altering peptide sequences or assembly conditions is important for versatility in bioengineering applications. Approaches to vary the mechanical properties of synthetic protein networks relies on the formation of covalent crosslinks, and increases in polymer concentration or control of assembly [ 110 , 134 , 137 , 138 , 139 , 140 ]. Hydrogelation of SAPs is crucial to create a mimic of the highly hydrated and bioactive ECM scaffold.…”

“…SAPs can also be blended to form co-assembled hydrogels [ 110 , 139 , 140 , 162 ]. To achieve this, peptides are combined before assembly, allowing for the formation of complex networks consisting of fibrils of mixed sequence.…”

The Body’s Cellular and Molecular Response to Protein-Coated Medical Device Implants: A Review Focused on Fibronectin and BMP Proteins

“…The colocalized synthetic oligopeptide of RGD and its synergistic PHSRN motifs in a poly(ethyl glycol) hydrogel improved osteoblast adhesion, spreading, and focal contact formation [ 43 ]. The co-assembly of two self-assembling peptides, RGD, and PHSRN, that produced a self-supporting hydrogel also exhibits synergistic effects in adhesion, spreading, and proliferation in vitro compared to RGD alone [ 42 ]. Further, the finish of an implant surface, whether it is smooth and polished or rough, is important for RGD coating effectiveness at osteointegration.…”

“…[ [41][42][43][44][45][46][47] FN recombinant fragment Enhanced cell adhesion and/or downstream osteoblastic differentiation by integrin-specific binding domains, e.g., FNIII7-10, FNIII9-10.…”