Nano-patterned, mechano-tunable, ECM mimetic substrates for cell adhesion studies under strain

Adherent cells are sensitive to physical and chemical cues in their environment and can adapt their response accordingly. Receptors in cells membrane are crucial elements in the recognition of such signals by binding external ligands (key-and-lock principle). Thereby these receptors and associated proteins transduce external cues in internal signals. Chemical nature of the ligands and their spatial arrangement can provide important information to the cells. In particular, spatial clustering of the ligands at the nanometer scale is considered to be a general principle by which the signal transduction of many biological processes gets modulated. Examples are the formation of focal adhesions (FAs) or of the immunological synapse. To investigate such clustering effects at the molecular scale, there is an immanent need for the precise chemical modification of surfaces. Therefore, the aim of this work was to (i) develop a substrate on which the spatial arrangement of bound particles could be precisely controlled and varied at the nanometer scale, and (ii) to demonstrate the utility of such substrates in cell adhesion studies. During this work an elastic poly(ethyleneglycol)-diacrylate (PEG-DA) hydrogel (HG) was used as carrier substrate on which an array of gold nano-particles (AuNPs) with well defined inter-particle distances (Delta L) was immobilized. Delta L could be successfully varied at the nanometer scale by mechanical stretching of the carrier substrate. For cell adhesion studies the AuNPs were functionalized with a c(RGDfK) peptide so that integrin binding was preferentially invoked. Due to the protein repellent properties of PEG, the AuNPs constituted the only anchor points on which cells could adhere. Thereby nanometer precision on the spatial arrangement of the ligands was achieved. To show the versatility of PEG-DA HGs as bio-mimetic substrates, the surfaces of a series of HGs were homogeneously functionalized with Fibronectin (FN). Strain applied to the HGs was successfully transmitted to cells and FAs and their reaction to the strain and to the change in inter-ligand distances was monitored and analyzed. The anisotropy generated in the ligand array by the uni-axial stretching influenced cell adhesion. FAs reaction was more prominent when stretched on HGs + FN than on HGs + AuNPs + RGD. In summary, fabrication of a material system for the dynamic variation of Delta L in the tens of nanometers was accomplished in this work. By rendering these substrates bio-mimetic, cell adhesion studies with dynamic variation of inter-ligand distances could be performed