**3. Conclusion**

408 Biomaterials – Physics and Chemistry

observed to adsorb to virtually any surface, and because FN is generally considered to be the most important cell adhesion mediating protein in serum and consequently has been

Table 1. Protein adsorption to the surface of the (topographically patterned) PEG-based hydrogel; qualitative results are given for samples that were incubated with serum (100% or 10% in buffer solution) or with buffer solutions containing a mixture of the pure proteins Vitronectin (VN) and Fibronectin (FN) in various ratios. Reproduced from: Schulte et al.,

Fig. 10. Cell adhesion on PEG hydrogels enabled by surface topography and/or preincubation with the cell adhesion-mediating protein VN. Reproduced from: Schulte et al.,

In order to verify whether this small but significant amount of adsorption of VN to the PEG surface is responsible for the observed cell adhesion on topographically patterned hydrogels we investigated the pre-incubated hydrogels (both smooth and patterned) in cell culture.

*Macromol. Biosci. (in press)*. Copyright 2011 John Wiley and Sons.

(2011) *Macromol. Biosci.* (*in press).* Copyright 2011 John Wiley and Sons.

much more studied than VN.

Hydrogels are of high relevance for several biomedical applications. We have described the fabrication of a hydrogel system based on poly(ethylene glycol) and evaluated the potential of this PEG-based gel as a patternable biomaterial. PEG-based polymers are of great importance as biomaterials for applications in cell and tissue engineering, as coating of implants or biosensors, and as drug delivery systems. In particular, PEG coatings have been used to minimize surface biofouling by plasma proteins to create surfaces that are "invisible" to cells. Cell biological studies with murine fibroblasts (NIH L929) confirmed the expected non-adhesive nature of the smooth hydrogel surfaces and furthermore ruled out any toxic effect of the material. Alterations of the mechanical properties could easily be achieved by varying the crosslinking density.

The most striking result from our studies is that the very popular and versatile PEG biomaterial is not cell-repellent per se. Only when the surface of the bulk PEG hydrogels is smooth it is anti-adhesive to cells, and this applies to all hydrogels we have investigated with a stiffness ranging from 0.1 to 1 MPa. However, we have discovered that on the same PEG hydrogels when decorated with micropatterns of topography, cells are able to adhere and spread. We have explored several underlying biochemical, biophysical and biomechanical factors that could attribute to this phenomenon and found that these factors do have an effect indeed, and notably the combination of these parameters, e.g. protein adsorption, surface topography and substrate compliance, work together to enable cell adhesion to the intrinsically anti-adhesive PEG biomaterial.

More specifically, three investigated PEG-based hydrogels with different stiffness were all cell anti-adhesive when smooth. However, in combination with topography, the softer gels were clearly more attractive for the cells; on softer gels with the same pattern geometry, significantly more cells adhered and spread than on the intermediate or stiffer gels. It seems that the compliance of the softer gels enables the cells to 'squeeze' into the grooves, although the cells apparently deform their own cytoskeleton rather than the topographic features.

We also discovered that a slight but significant amount of the ECM-protein Vitronectin is able to adsorb to the PEG surface and that this leads to an increase in initial cell adhesion during the first 4 hours of cell culture. However, this effect rapidly falls off. The effect of

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topography is a more durable effect that dominates at longer time scales, suggesting its role in the enabling of stable adhesion complexes, which is a process that occurs during more than several hours. We consider that the topographic features may provide shelter to the cells and prolong their residence time inside of the grooves. Added to this, the 'pulling' of the cells on the weakly bound cell adhesion proteins may be less effective when they are confined between the vertical walls than when the surface is smooth; as a consequence focal adhesion contacts can develop into stable focal adhesion complexes.

Thus, the combination of different (bio)chemical, physical and mechanical properties of the PEG hydrogels results in the observed cell adhesion on this intrinsically anti-adhesive biomaterial. The effects are difficult to disentangle, and marked synergistic effects were observed for example when using topographically patterned hydrogels that were incubated with Vitronectin prior to cell culture.

As PEG is generally well known for its anti-adhesive properties and is widely applied in biomedical applications, it is important to take into consideration what our study has shown: physical and mechanical surface properties can impede the anti-adhesive characteristics of PEG. On the other hand it also opens new opportunities for biomimetic material design which does not rely on complicated and expensive biochemical surface functionalization for manipulating cellular responses.
