Quantitative control of protein and cell interaction with nanostructured surfaces by cluster assembling

The development of smart prosthetics, scaffolds, and biomaterials for tissue engineering and organ-on-a-chip devices heavily depends on the understanding and control of biotic/abiotic interfaces. In recent years, the nanometer scale emerged as the predominant dimension for processes impacting on protein adsorption and cellular responses on surfaces. In this context, the extracellular matrix (ECM) can be seen as the prototype for an intricate natural structure assembled by nanoscale building blocks forming highly variable nanoscale configurations, dictating cellular behavior and fate. How exactly the ECM nanotopography influences mechanotransduction, that is, the cellular capacity to convert information received from the ECM into appropriate responses, remains partially understood due to the complexity of the involved biological structures, limiting also the attempts to artificially reproduce the nanoscale complexity of the ECM. In this Account, we describe and discuss our strategies for the development of an efficient and large-scale bottom-up approach to fabricate surfaces with multiscale controlled disorder as substrates to study quantitatively the effect of nanoscale topography on biological entities. Our method is based on the use of supersonic cluster beam deposition (SCBD) to assemble, on a substrate, neutral clusters produced in the gas phase and accelerated by a supersonic expansion. The assembling of clusters in the ballistic deposition regime follows simple scaling laws, allowing the quantitative control of surface roughness and asperity layout over large areas. Due to their biocompatibility, we focused on transition metal oxide nanostructured surfaces assembled by titania and zirconia clusters. We demonstrated the engineering of structural and functional properties of the cluster-assembled surfaces with high relevance for interactions at the biotic/abiotic interface. We observed that isoelectric point and wettability, crucial parameters for the adhesion of biological entities on surfaces, are strongly influenced and controlled by the nanoscale roughness. By developing a high-throughput method (protein surface interaction microarray, PSIM), we characterized quantitatively the capacity of the nanostructured surfaces to adsorb proteins, showing that with increasing roughness the adsorption rises beyond what could be expected by the increase in specific area, paralleled by an almost linear decrease in protein binding affinity. We also determined that the spatial layout of the surface asperities effectively perceived by the cells mimics at the nanoscale the topographical ECM characteristics. The interaction with these features consequently regulates parameters significant for cell adhesion and mechanotransductive signaling, such as integrin clustering, focal adhesion maturation, and the correlated cellular mechanobiology, eventually impacting the cellular program and differentiation, as we specifically showed for neuronal cells.