Hydrogel-fibre composites with independent control over cell adhesion to gel and fibres as an integral approach towards a biomimetic artificial ECM.

In the body, cells are surrounded by an interconnected mesh of insoluble, bioactive protein fibres to which they adhere in a well-controlled manner, embedded in a hydrogel-like highly hydrated matrix. True morphological and biochemical mimicry of this so-called extracellular matrix (ECM) remains a challenge but appears decisive for a successful design of biomimetic three-dimensional in vitro cell culture systems. Herein, an approach is presented which describes the fabrication and in vitro assessment of an artificial ECM which contains two major components, i.e. specifically biofunctionalized fibres and a semi-synthetic hyaluronic acid-based hydrogel, which allows control over cell adhesion towards both components. As proof of principle for the control of cell adhesion, RGD as well-known cell adhesive cue and the control sequence RGE are immobilized in the system. In vitro studies with primary human dermal fibroblasts were conducted to evaluate the specificity of cell adhesion and the potential of the composite system to support cell growth. Finally, one possible application example for guided cell growth is shown by the use of oriented fibres in a hydrogel matrix.

Microengineered PEG hydrogels: 3D scaffolds for guided cell growth.

Designing three-dimensional (3D) scaffolds for selective manipulation of cell growth is of high relevance for applications in regenerative medicine. Especially, scaffolds with oriented morphologies bear high potential to guide the restoration of specific tissues. The fabrication of hydrogel scaffolds that support long-term survival, proliferation, and unidirectional growth of embedded cells is presented here. Parallel channel structures are introduced into the bulk hydrogels by uniaxial freezing, providing stable, and uniform porosity suitable for cell invasion (pore diameters of 5-15 µm). In vitro assessment of the scaffolds with murine fibroblasts (NIH L929) shows a remarkable unidirectional movement along the channels, with the cells traveling several millimeters through the hydrogel.

Development and characterization of a coronary polylactic acid stent prototype generated by selective laser melting.

In-stent restenosis is still an important issue and stent thrombosis is an unresolved risk after coronary intervention. Biodegradable stents would provide initial scaffolding of the stenosed segment and disappear subsequently. The additive manufacturing technology Selective Laser Melting (SLM) enables rapid, parallel, and raw material saving generation of complex 3- dimensional structures with extensive geometric freedom and is currently in use in orthopedic or dental applications. Here, SLM process parameters were adapted for poly-L-lactid acid (PLLA) and PLLA-co-poly-ε-caprolactone (PCL) powders to generate degradable coronary stent prototypes. Biocompatibility of both polymers was evidenced by assessment of cell morphology and of metabolic and adhesive activity at direct and indirect contact with human coronary artery smooth muscle cells, umbilical vein endothelial cells, and endothelial progenitor cells. γ-sterilization was demonstrated to guarantee safety of SLM-processed parts. From PLLA and PCL, stent prototypes were successfully generated and post-processing by spray- and dip-coating proved to thoroughly smoothen stent surfaces. In conclusion, for the first time, biodegradable polymers and the SLM technique were combined for the manufacturing of customized biodegradable coronary artery stent prototypes. SLM is advocated for the development of biodegradable coronary PLLA and PCL stents, potentially optimized for future bifurcation applications.

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.

Combined influence of substrate stiffness and surface topography on the antiadhesive properties of Acr-sP(EO-stat-PO) hydrogels.

Biomaterials that prevent nonspecific protein adsorption and cell adhesion are of high relevance for diverse applications in tissue engineering and diagnostics. One of the most widely applied materials for this purpose is Poly(ethylene glycol) (PEG). We have investigated how micrometer line topography and substrate elasticity act upon the antiadhesive properties of PEG-based hydrogels. In our studies we apply bulk hydrogel cross-linked from star-shaped poly(ethylene oxide-stat-propylene oxide) macromonomers. Substrate surfaces were topographically patterned via replica molding. Additionally, the mechanical properties were altered by variations in the cross-linking density. Surface patterns with dimensions in the range of the cells' own size, namely 10 µm wide grooves, induced significant cell adhesion and spreading on the Acr-sP(EO-stat-PO) hydrogels. In contrast, there was only little adhesion to smaller and larger pattern sizes and no adhesion at all on the smooth substrates, regardless the rigidity of the gel. The effect of varied substrate stiffness on cell behavior was only manifest in combination with topography. Softer substrates with line patterns lead to significantly higher cell adhesion and spreading than stiff substrates. We conclude that the physical and mechanical surface characteristics can eliminate the nonadhesive properties of PEG-based hydrogels to a large extent. This has to be taken into account when designing surfaces for biomedical application such as scaffolds for tissue engineering which rely on the inertness of PEG.

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)ß(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.

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.

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.