Fabrication Of Nanostructured ECM-Mimetic Interfaces For Control Of Cell Adhesion Behaviors

Cellular responses depend strongly on the chemo-mechanical features of underlying interfaces. In particular, surface nanofeature of tissue-repairing materials is a cruicial aspect which governs cellular function and healing response. Emerging studies have developed various materials coupled with cell-adhesive ligands such as the peptide of arginine-glycine-aspartic acid (RGD) to mimic extracellular matrix (ECM) and to regulate cellular behaviors. The nanoscale organization of RGD ligands on nonfouling background affords a valuable tool to study cellular responses to ECM motifs on the molecular level. So far, is has been revealed that the interfacial arrangement and specificity of ligands exert strong effects on cell fate. However, there has been, to date, no report comparing cellular responses to nanostructured surfaces with ordered and disordered organization of RGD ligands. Especially, how cells sense the order and disorder of ligand organization in ECM environment still remains a key question.The aim of this work is to present novel platforms of well-controlled ordered and disordered nanopatterns positioned with a cyclic peptide of arginine-glycine-aspartic acid-D-phenyl alanine-lysine [c(RGDfK)] on a bioinert poly(ethylene glycol) (PEG) background, to study the critical issue that how the nanoscopic spatial patterning of the integrin-specific ligands influences cell adhesion. This is the first time that the nanoscale order of RGD ligand patterns was varied quantitatively, and tested for its impact on the adhesion of tissue cells. For primarily testing how the mechanical property of bulk material affects cellular responses, both rigid and soft background materials mimicking ECM was designed.As the first step of this work, well-controlled parallel ordered and disordered gold (Au) nanopatterns with a series of average interparticle distances and varied orders under a defined density on inorganic substrates (glass or Si wafers) were fabricated via the newly-developed block copolymer micelle nanolithography (BCMN) technique with or without addition of order-interfering reagents as mentioned in this work. Various experimental conditions were systematically examined for modulating the key parameters of Au nanopatterns. Besides, new method and software were developed for statistical analysis of nanopatterns.In the next part of this work, we generated nanopatterns of cell adhesive ligand and performed corresponding cell experiments. Structuring c(RGDfK) ligand nanopatterns on nonfouling rigid background was achieved by PEG-silane passivation technique combined with c(RGDfK) peptide coupling. Both ordered and disordered nanopatterns of c(RGDfK) ligand on PEG-passivated supports were successfully fabricated, by which we controlled the lateral positioning of integrins as single adhesion receptors in defined orders and spacings in cell adhesion processes. Based on the cell adhesion assays in a comparative manner, our findings reveal that integrin clustering is dependent on the local order of ligand arrangement when the global average ligand spacing is larger than-70 nm. By decoupling RGD average density and molecularly defined local interligand distances, this work confirmed the existence of a critical local interligand distance of-70 nm, above which cell adhesion was strongly reduced. Stable focal adhesion of cells on disordered nanopattern surfaces is activated at less global average ligand density than on ordered ones. The related mechanism of the phenomenon was discussed as well.Thirdly, c(RGDfK) nanopattems were constructed on soft polymeric materials such as PEG-DA hydrogel by newly-established transfer lithography technique combined with peptide functionalization. The mechanical property of PEG-DA hydrogel can be well controlled by adjusting the crosslinking density of the polymer network. Cryo-SEM microscopy was used for visualizing the PEG hydrogel surfaces positioned with various nanopatterns. Based on this platform, primary cell experiments were carried out to detect how cells interact with the nanopatterned hydrogels with tunable viscoelasticity and varied organization of c(RGDfK) ligands at nanoscle.Overall, the innovative nanostructured interfaces mimicking ECM developed in the present work serve as a versatile model to study cellular behaviors. The novel material techniques encourage one to extensively investigate the cell-material crosstalk on the molecular level, as well as to design new nanomaterials for regenerative medicine.