Introduction: In the body cells are surrounded by a complex arrangement of proteins and biomolecules called the extracellular matrix (ECM). Attempts to recreate this environment in vitro have yielded broadly applicable strategies, such as including the RGD tri-peptide for cell adhesion, but attempts to recreate the complex arrangement of ligands for controlled cell signaling have been more difficult to achieve. In vivo, a single cell surface protein can be bound simultaneously by several epitopes. For instance, the a5b1 integrin synergistically binds both RGDS and PHSRN epitopes found approximately 3.5 nm apart from each other on the ECM protein fibronectin. This synergistic binding leads increases integrin specificity and has been shown to induce osteogenesis in human mesenchymal stromal cells (hMSCs). Here we designed self-assembled peptide hydrogels that are modified to display and RGDS and PHSRN epitopes at specific locations along the peptide. Methods and Materials: Peptides were synthesized using a combination of solid phase peptide synthesis and click chemistry. Peptides with the bioactive epitopes (RGDS and PHSRN) at spacings of 0.6nm, 3.5 nm and 5.5 nm, as well as RGDS-only and PHSRN-only controls were mixed 1:50 with unlabeled peptides. These were dissolved in water at 4 weight%, mixed 1:1 with cells in media and gelled with the addition of 20 mM CaCl2. Human umbilical vein endothelial cells (HUVECs) were seeded into the gels and studied at 4,8 and 24 hours for integrin gene expression and immunostaining. Results and Discussion: We designed a self-assembling peptide hydrogel using b-sheet forming peptide with alternating hydrophilic-hydrophobic amino acid residues. We then mixed in peptides were then selectively modified to display RGDS and PHSRN side chains at desired distances using a combination of amide and click chemistry. Since b-sheets are rigid in solution, the distance between any two amino acids along the backbone can be determined a priori and spaced at 0.6 nm increments. This backbone self-assembles into one-dimensional, twisted nanostructures with diameters around 5 nm and lengths on the micron scale based on scanning and transmission electron microscopy. This geometry suggests these nanostructures are one peptide in diameter and only a few b-sheets thick, with the b-sheets aligned with the long axis of the fiber. The presence of b-sheets was confirmed using circular dichroism. HUVECs were seeded into the gels and their spreading and integrin gene expression was monitored to assess the impact that epitope spacing has on cell behavior. Polymerase chain reaction (PCR) showed an upregulation of the a5 integrin subunit at 4 hours, and immunocytochemistry showed both an increase in cell spreading and an increase a5b1 integrin straining in gels that had the RGDS and PHSRN epitopes at the correct distance. Conclusion: The body uses exquisite fidelity in not only controlling the expression of proteins and biomolecules, but also their location and spacing in the cell and extracellular matrix. By using the known geometry present in peptide b-sheets we were able to controllably space RGDS and PHSRN epitopes the 3.5 nm apart they were found in fibronectin. This spacing caused an increase in cells spreading and a5b1 integrin expression compared to hydrogels that displayed both epitopes at non-physiological spacings. Designing biomaterials that not only include the biological epitopes found in the ECM but also mimic their native presentation is important in creating matrices that recapitulate the native environment for improved cell signaling.
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