Mar Diez

Surface topography induces fibroblast adhesion on intrinsically nonadhesive poly(ethylene glycol) substrates.

Important in developing new biomaterials is the prevention of unspecific protein adsorption and cell interactions that in vivo can lead to a foreign body reaction. On the other hand, the material should support the growth of a specific cell type in a defined way. We investigate the possibility of manipulating cellular behavior on an intrinsically nonadhesive material by topographic patterning without additional surface chemistry modifications. The biomaterial applied is a hydrogel cross-linked from star-shaped poly(ethylene glycol) macromonomers (starPEG). Cell biological studies with a mouse fibroblast cell line (L929) showed that, while substrates with a smooth surface are nonadhesive, as expected, imprinted topography enabled cell adhesion and spreading. The fibroblasts aligned to micrometer groove patterns and were, depending on the respective dimensions, able to span or enter the grooves. Especially substrates with topography dimensions in the cell size range or smaller (<10 microm) lead to an establishment of stable cell-surface contacts (vinculin and actin accumulation). On micrometer post patterns the cells spread on top of the pillars and wrapped around the structures. The strong influence of the topography shows that nonadhesive materials do not necessarily have to be specifically biofunctionalized to enable cell adhesion. Possible explanations for the peculiar cell behavior are discussed in terms of (initial) protein adsorption and geometry-dependent cytoskeletal arrangements.

Induction of specific macrophage subtypes by defined micro-patterned structures.

In this study, we investigated the influence of different perfluoropolyether (PFPE) microstructures on the inflammatory response of human macrophages. We generated four different microstructured PFPE surfaces by replica molding from silicon masters. The function-associated surface markers 27E10 and CD163 were monitored using flow cytometry to measure the pro- and anti-inflammatory reactions. Inflammatory mediator expression was measured at the protein and mRNA level. Lipopolysaccharide treatment served as positive control for pro-inflammatory activation. We observed that each micropattern induced a specific morphology, phenotype and mediator profile. A microstructure of regular grooves induced a pro-inflammatory phenotype (M1) which was not accompanied by release of pro-inflammatory mediators. However, the larger cylindrical posts induced an anti-inflammatory phenotype (M2) with a remarkable down-regulation of CXCL10. Smaller posts with a shorter distance exhibited a stronger pro-inflammatory response than those with a longer distance, on the levels of both phenotype and mediator release. Regression analysis suggests that the geometrical parameters of the microstructures, specifically the period of structures, may play an important role in macrophage response. Optimization of such microstructures may provide a method to invoke a predictable response of macrophages to implants and control the mediator release.

Deformation of nanostructures on polymer molds during soft UV nanoimprint lithography

Soft nanoimprint lithography (soft NIL) relies on a mechanical deformation of a resist by a patterned polymer used as a mold. Here, we report on the investigation of the nanopattern fidelity of the high pressure imprint process based on a perfluorinated polyether (PFPE) soft mold material. The perfluorinated polyether material was found to be well suited to transfer the mold pattern into the resist by a direct imprinting process at low cost. Moderate deformations of the polymer mold structures occurring during the high pressure imprint are systematically studied. Features of decreased size are found to be more sensitive to pattern distortions. An optimized pattern design with increased structure density and constant pattern ratio is developed to minimize deformation effects. Imprints performed on the basis of these design rules result in reduced deformations and repeal their size dependence. The improved pattern transfer, especially for small structural elements, turns the direct and cost-effective soft UV-NIL into an interesting technique also for patterning tasks in the lower nanometer range.

Nanomolding of PEG‐Based Hydrogels with Sub‐10‐nm Resolution

A simple, soft nanolithographic method is used to fabricate sub-10-nm structures on star polyethylene glycol-based hydrogels and perfluoropolyether-based materials. Very small features, for example, gold nanoparticles of size approximately 8 nm with an interparticle distance of approximately 100 nm, are successfully reproduced from a hard silicon master into both elastomers. Scanning force microscopy is used to investigate the replicas, and the original hexagonal pattern of the nanoparticles is clearly recognized. In addition, both replicas are usable as secondary, soft molds yielding positive copies of the primary, hard master. The results presented here show similar replication capabilities for both elastomers despite the markedly different properties of the precursors. Moreover, the hydrogel material can be easily peeled off from both soft and silicon masters without the need for surface treatment. The procedure allows nanopatterning of a biocompatible material over large areas, which is a useful tool to investigate cellular responses to defined nanotopography.

A hydrophobic perfluoropolyether elastomer as a patternable biomaterial for cell culture and tissue engineering

We present a systematic study of a perfluoropolyether (PFPE)-based elastomer as a new biomaterial. Besides its excellent long-term stability and inertness, PFPE can be decorated with topographical surface structures by replica molding. Micrometer-sized pillar structures led to considerably different cell morphology of fibroblasts. Although PFPE is a very hydrophobic material we could show that PFPE substrates allow cell adhesion and spreading of primary human fibroblasts (HDF) very similar to that observed on standard cell culture substrates. Less advanced cell spreading was observed for L929 (murine fibroblast cell line) cells during the first 5 h in culture which was accompanied by retarded recruitment of α(v)β(3)-integrin into focal adhesions (FAs). After 24 h distinct FAs were evident also in L929 cells on PFPE. Furthermore, organization of soluble FN into a fibrillar ECM network was shown for hdF and L929 cells. Based on these results PFPE is believed to be a suitable substrate for several biological applications. On the one hand it is an ideal cell culture substrate for fundamental research of substrate-independent adhesion signaling due to its different characteristics (e.g. wettability, elasticity) compared to glass or TCPS. On the other hand it could be a promising implant material, especially due to its straightforward patternability, which is a tool to direct cell growth and differentiation.

Topography-Induced Cell Adhesion to Acr-sP(EO-stat-PO) Hydrogels: The Role of Protein Adsorption

Topographic surface patterning of intrinsically non-adhesive P(EO-stat-PO)-based hydrogels can lead to the adhesion and spreading of fibroblasts. Explanations for this unexpected behavior are discussed, particularly with regard to non-specific protein adsorption from the serum-supplemented culture medium. The presence of serum proteins is shown to be essential for adhesion. Adsorption of plasma and ECM proteins (Fibronectin (FN) and Vitronectin (VN)) to the hydrogels is possible. The effect of VN on initial cell adhesion is analyzed in detail. It appears that VN is the main serum component that is crucial for initial cell adhesion to PEG and that surface topography is essential for further, durable adhesion establishment, and spreading.

Cell Adhesion and Spreading on an Intrinsically Anti-Adhesive PEG Biomaterial

This Chapter deals with bulk hydrogels consisting of a widely used biomaterial: poly(ethylene) glycol (PEG). PEG is renown for its bio-inertness; it is very effective in suppressing non-specific protein adsorption (NSPA) and thereby preventing cell adhesion. However, we have observed unexpected adhesion of fibroblast cells to the surface of bulk PEG hydrogels when the surface was decorated with micrometer-sized, topographic patterns. This Chapter describes the aim of our investigations to unravel the biophysical, biochemical and biomechanical reasons why these cells do adhere to the intrinsically antiadhesive PEG material when it is topographically patterned.