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Control over cell-material surface interactions is the key to many new and improved biomedical devices. It can only be achieved if interactions that are mediated by nonspecifically adsorbed serum proteins are minimized and if cells instead respond to specific ligand molecules presented on the surface. Here, we present a simple yet effective surface modification method that allows for the covalent coupling and presentation of specific biological signals on coatings which have significantly reduced nonspecific biointerfacial interactions. To achieve this we synthesized bottle brush type copolymers consisting of poly(ethylene glycol) methyl ether methacrylate and (meth)acrylates providing activated NHS ester groups as well as different spacer lengths between the NHS groups and the polymer backbone. Copolymers containing different molar ratios of these monomers were grafted to amine functionalized polystyrene cell culture substrates, followed by the covalent immobilization of the cyclic peptides cRGDfK and cRADfK using residual NHS groups. Polymers were characterized by GPC and NMR and surface modification steps were analyzed using XPS. The cellular response was evaluated using HeLa cell attachment experiments. The results showed strong correlations between the effectiveness of the control over biointerfacial interactions and the polymer architecture. They also demonstrate that optimized fully synthetic copolymer coatings, which can be applied to a wide range of substrate materials, provide excellent control over biointerfacial interactions. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

This study presents the easy and fast patterning of low molecular weight molecules that act as binding partners for proteins on Star PEG coatings. These coatings are prepared from isocyanate terminated star shaped prepolymers and form a highly cross-linked network on the substrate in which the stars are connected via urea groups and free amino groups are present. Streptavidin has been patterned on these layers by microcontact printing (muCP) of an amino reactive biotin derivative and consecutive binding of streptavidin to the biotin. Patterns of Ni(2+)-nitriltriacetic acid (NTA) receptors have been prepared by printing amino functional NTA molecules in freshly prepared Star PEG layers that still contain amino reactive isocyanate groups. Complexation of the NTA groups with Ni(II) ions enabled the binding of His-tag enhanced green fluorescent protein (EGFP) in the desired pattern on the substrates. Since the unmodified Star PEG layers prevent unspecific protein adsorption, His-EGFP could selectively be bound to the sample by immersion into crude, nonpurified His-tag EGFP containing cell lysate.

In this study we present the preparation of thin and ultrathin coatings from six-arm star-shaped isocyanate-terminated prepolymers on amino-functionalized silicon wafers. The backbone of the stars is a statistical copolymer of ethylene oxide and propylene oxide in the ratio 80:20 (Star PEG). Film preparation by spin coating from aqueous THF resulted in a variety of film morphologies that are determined by the water content of the solvent. Water is indispensable for activation of the isocyanate-terminated stars in solution and for proper cross-linking of the coatings on the substrate. This cross-linking results in a dense network of PEG chains on the substrate linked via urea groups with a mesh size of the network that corresponds to the arm length of the stars. Layer thickness variations between 3 and 500 nm revealed a strong dependence of the contact angle with water on the layer thickness which is explained by the chemical composition of the coatings. Due to the high functionality of the star-shaped prepolymers, free amino groups remain in the films that were detected by fluorescence microscopy after reaction with 4-chloro-7-nitrobenzofurazan (NBF). To test the system for the ability to prevent unspecific interaction with proteins, adsorption of fluorescence-labeled avidin was examined with fluorescence microscopy. For layer thicknesses between 3 and 50 nm, no protein adsorption could be detected.

Ordered surface nanostructures have attracted much attention in biotechnology and biomedical engineering because of their potential to modulate cell-surface interactions in a controllable manner. However, the ability to fabricate large area ordered nanostructures is limited because of high costs and low speed of fabrication. Here, we have fabricated ordered nanostructures with large surface areas (1.5 x 1.5 cm(2)) using a combination of facile techniques including colloidal self-assembly, colloidal lithography and glancing angle deposition (GLAD). Polystyrene (722 nm) colloids were self-assembled into a hexagonally close-packed (hcp) crystal array at the water-air interface, transferred on a biocompatible tantalum (Ta) surface and used as a mask to generate an ordered Ta pattern. The Ta was deposited by sputter coating through the crystal mask creating approximately 60-nm-high feature sizes. The feature size was further increased by approximately 200-nm-height respectively using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and osteogenic differentiation of primary human adipose-derived stem cells (hADSCs) were studied on these ordered nanostructures for up to 2 weeks. Our results suggested that cell spreading, focal adhesion formation, and filopodia extension of hADSCs were inhibited on the GLAD surfaces, while the growth rate was similar between each surface. Immunostaining for type I collagen (COL1) and osteocalcin (OC) showed that there was higher osteogenic components deposited on the GLAD surfaces compared to the Ta60 and FLAT surfaces after 1 week of osteogenic culture. After 2 weeks of osteogenic culture, alkaline phosphatase (ALP) activity and the amount of calcium was higher on the GLAD surfaces. In addition, osteoblast-like cells were confluent on Ta60 and FLAT surfaces, whereas the GLAD surfaces were not fully covered suggesting that the cell-cell interactions are stronger than cell-substrate interactions on GLAD surfaces. Visible extracellular matrix deposits decorated the porous surface can be found on the GLAD surfaces. Depth profiling of surface components using a new Ar cluster source and X-ray photoelectron spectroscopy (XPS) showed that deposited extracellular matrix on GLAD surfaces is rich in nitrogen. The fabricated ordered surface nanotopographies have potential to be applied in diverse fields, and demonstrate that the behavior of human stem cells can be directed on these ordered nanotopographies, providing new knowledge for applications in biomaterials and tissue engineering.

Background: The ability to present signalling molecules within a low fouling 3D environment that mimics the extracellular matrix is an important goal for a range of biomedical applications, both in vitro and in vivo. Cell responses can be triggered by non-specific protein interactions occurring on the surface of a biomaterial, which is an undesirable process when studying specific receptor-ligand interactions. It is therefore useful to present specific ligands of interest to cell surface receptors in a 3D environment that minimizes non-specific interactions with biomolecules, such as proteins. Conclusion: Overall, the studies presented an effective platform for the preparation of 3D scaffolds which contain effective conjugation sites for attachment of specific bioactive signals of interest, as well as actively reducing non-specific protein interactions. Method: In this study, surface-initiated atom transfer radical polymerization (SI-ATRP) of poly(ethylene glycol)-based monomers was carried out from the surface of electrospun fibers composed of a styrene/vinylbenzyl chloride copolymer. Surface initiated radical addition-fragmentation chain transfer (SI-RAFT) polymerisation was also carried out to generate bottle brush copolymer coatings consisting of poly(acrylic acid) and poly(acrylamide). These were grown from surface trithiocarbonate groups generated from the chloromethyl styrene moieties existing in the original synthesised polymer. XPS was used to characterise the surface composition of the fibers after grafting and after coupling with fluorine functional XPS labels. Results: Bottle brush type coatings were able to be produced by ATRP which consisted of poly(ethylene glycol) methacrylate and a terminal alkyne-functionalised monomer. The ATRP coatings showed reduced non-specific protein adsorption, as a result of effective PEG incorporation and pendant alkynes groups existing as part of the brushes allowed for further conjugation of via azide-alkyne Huisgen 1,3-dipolar cycloaddition. In the case of RAFT, carboxylic acid moieties were effectively coupled to an amine label via amide bond formation. In each case XPS analysis demonstrated that covalent immobilisation had effectively taken place. Refereed/Peer-reviewed