Large area protein patterning reveals nanoscale control of focal adhesion development.

Focal adhesion development in cells adherent to surface bound fibronectin presented as 200, 500, or 1000 nm diameter circular patches or as homogeneous controls is studied by fluorescence and scanning electron microscopy. Fundamental cellular processes such as adhesion, spreading, focal adhesion and stress fiber formation are shown to be dependent on the spatial distribution of ligands at this scale. Large area samples enable the study of whole cell populations and opens for new potential applications.

Polydopamine/liposome coatings and their interaction with myoblast cells.

Surface-mediated drug delivery is a recent concept, where active surface coatings are employed to deliver therapeutic cargo to cells. Herein, we explore the potential of liposomes embedded in polydopamine (PDA) coatings to serve as drug deposits stored on planar substrates. We quantify the PDA growth rate on glass by XPS and show that PDA coatings support myoblast adherence and proliferation. Further, PDA capping layers were deposited on glass substrates precoated with poly(L-lysine) and zwitterionic liposomes. Already thin PDA capping layers render liposome coated surfaces cell adhesive. We experimentally show for the first time, the internalization of a model hydrophobic cargo, that is, fluorescent lipids embedded within the lipid bilayer of liposomes by the cells from the surface. This is evident from the fluorescence exhibited by the cells grown on PDA coatings containing fluorescently labeled liposomes, with the highest fluorescent intensity found in the close proximity of the cell nuclei. The cargo uptake efficiency depends on the thickness of the PDA capping layer and the cell residence time on the coated substrates. Taken together, we demonstrate the first step toward the establishment of a versatile approach using liposomal drug deposits in polymer thin films for surface-mediated drug delivery.

A combinatorial screening of human fibroblast responses on micro-structured surfaces.

Biomaterial surfaces structured with topographical features have been predicted to play an important role in the next generation of biomedical implants. Specific trends with regard to the influence of the topographical effect on cellular behavior are however challenging to establish due to differences in the topographical features and geometries in the various studies. Here, we demonstrate the use of a highly versatile combinatorial screening approach to identify the effect of 169 distinct surface topographies, consisting of pillars, on fibroblast proliferation and mechanical response. Altering the inter-pillar gap size of the structures revealed a significant change in fibroblast proliferation and identified obvious stress-induced changes in the cytoskeleton and focal adhesion morphology. Larger (4-6 µm) inter-pillar gap sizes reduced fibroblast proliferation and elicited a strong elongation leading to a disruption of the actin cytoskeleton anchored primarily to focal adhesions located between the pillars. Smaller (1-2 µm) inter-pillar gap sizes, on the contrary, caused the fibroblasts to proliferate comparable to cells on a non-structured surface with cells having a clear actin cytoskeleton attached to focal adhesions located mostly on top of the pillars. The approach reveals a strong correlation between the exact topographical periodicities and cellular responses such as cell proliferation, cell morphology and focal adhesion.

Osteopontin presentation affects cell adhesion-Influence of underlying surface chemistry and nanopatterning of osteopontin.

Osteopontin is a promising coating material for biomaterials, being important both in remodeling and formation of mineralized tissue and in immunological responses. We have investigated cell attachment to osteopontin adsorbed at different surface chemistries (-NH(2), -COOH, -CH(3), and bare gold) and to osteopontin presented as a nanopattern of 50 nm protein patches separated by a nonadhesive background. MDA-MB-435 cells adhere well to osteopontin presented at the hydrophilic chemistries (-NH2, -COOH, and gold) suggesting that osteopontin is presented in a functional form on these surfaces. On the amine surface, the cell attachment appears partly driven by electrostatic attraction between the positively charged substrate and the negatively charged cell membrane, whereas the spreading of the cells depends on the specific interaction with osteopontin presented at the surface. Significantly, fewer cells adhere to osteopontin presented at the methyl-terminated hydrophobic surface and the cells are less spread. On the nanopatterned osteopontin, only a very low number of cells adhered and those few attached cells showed an elongated morphology with few adhesion points to the surface. This indicates that the adhesive patches are not large enough to support stable focal contacts. The good cell attachment and spreading on the hydrophilic surfaces holds promise for osteopontin as a future coating for biomaterials.

Interaction of human mesenchymal stem cells with osteopontin coated hydroxyapatite surfaces.

In vitro studies of the initial attachment, spreading and motility of human bone mesenchymal stem cells have been carried out on bovine osteopontin (OPN) coated hydroxyapatite (HA) and gold (Au) model surfaces. The adsorption of OPN extracted from bovine milk was monitored by the quartz crystal microbalance with dissipation (QCM-D) and the ellipsometry techniques, and the OPN coated surfaces were further investigated by antigen-antibody interaction. It is shown that the OPN surface mass density is significantly lower and that the number of antibodies binding to the resulting OPN layers is significantly higher on the HA as compared to the Au surfaces. The initial attachment, spreading and motility of human mesenchymal stem cells show a larger cell area, a faster arrangement of vinculin in the basal cell membrane and more motile cells on the OPN coated HA surfaces as compared to the OPN coated Au surfaces and to the uncoated Au and HA surfaces. These in vitro results indicate that there may be great potential for OPN coated biomaterials, for instance as functional protein coatings or drug delivery systems on orthopaedic implants or scaffolds for tissue-engineering.