Mimicking the Cellular Environment: Effects of Elastic Nanopatterned Substrates on Integrin-Mediated Cellular Interactions

Abstract

An artificial substrate system, according to the biophysical and biochemical properties of the extracellular matrix in connective tissues, has been developed. The Young's moduli EY of poly(ethylene glycol)-diacrylate (PEG-DA) based hydrogel substrates span more than four orders of magnitude between 0,6kPa and 6MPa. Since PEG-DA substrates are protein repellent, they were decorated by extended gold nanoparticle arrays, manufactured by block copolymer micellar nanolithography. To provide bioactivity in terms of cell adhesion c(RGDfK) peptide, which is specific for αVβ3 integrins, was immobilized on the nanoparticles. The interparticle spacing and, hence, spacing of integrin binding sites ΔL could be precisely tuned, independently of the substrate rigidity between 20nm and 160nm. This system was used to investigate the behavior of fibroblasts as a function of changes within two-dimensional parameters space ΔL;EY). To this end, cell spreading area and cell-substrate interaction forces were determined by phase contrast microscopy and single cell force spectroscopy (SCFS), respectively. First, the effect of variation of ligand spacing on cellular behavior was investigated on hard substrates (EY>100kPa). We could demonstrate a strong increase in detachment force and spreading area on substrates featuring low ligand spacing. Than, substrate compliance was tuned whereas the ligand spacing was kept at approximately 50nm. This reveals a significant decrease in spreading area and detachment force on soft substrates (EY<8kPa). Additionally, both environmental parameters were varied simultaneously. Results from these experiments were determined as a function of hydrogel stiffness and integrin ligand distance. They revealed two tactile set points, thresholds in cellular sensing behavior, at EY=8kPa and ΔL=70nm, after 6, 12, and 24 hours of adhesion. Moreover, according to the hierarchical phase model in cellular behavior, elasticity was identified to be the dominant parameter in cellular sensing processes.