**2.3 Fibroblast culture on smooth PEG hydrogel substrates**

In order to assess the cytocompatibility of the PEG-based hydrogels in direct contact with cells, L929 cells were cultured directly on the surface of bulk free-standing substrates. Samples of the three introduced elasticities were applied and cell morphology was documented by live imaging after 24 h and 48 h in culture (**Figure 5**). Subsequently, fixed and dried cells were further evaluated by means of electron microscopy (FESEM).

As can be seen in Figure 5, the cell morphology on the three different PEG substrates did not vary significantly, all cells displayed a round shape and only little or no stable cell adhesion was evident. This clearly confirms the cell-repulsive properties of the PEGbased material. Electron microscopy further showed that the rinsing and fixation of the samples led to the removal of a large number of cells. This observation underlines that only a negligible amount of cells was able to establish a contact to the surface. The majority of the cells tended to form aggregates after longer cultivation times. The number of cells visible on the images of the different samples cannot be compared directly as a slight shaking of the medium led to an immediate re-destribution of the cells above the surface.

Based on the observation that the cells were not able to build stable contacts to the surface the consequences for intracellular processes were studied as well. Many anchoragedependent cell types stop to proliferate or can undergo a programmed cell death (apoptosis) without the presence of integrin-mediated cell-surface contacts (Frisch & Francis, 1994; Gilmore, 2005). The amount of the apoptotic markers caspase-3 and caspase-7 after 48 h of cultivation time on the three different PEG substrates and polystyrene (PS) was assessed with a commercially available assay and compared to a culture where apoptosis was specifically induced by staurosporin addition (values set to 100 %). The results are depicted in **Figure 6**.

As seen in Figure 6, cells cultured on the three different sP(EO-stat-PO) hydrogels did not show an enhanced level of apoptotic activity compared to those seeded on the control substrate PS. This observation is in accordance with results from other groups showing that fibroblasts are not very sensitive to lack of adhesions to a solid substrate if serum is present in the medium (Ishizaki et al., 1995; McGill et al., 1997). There was also no significant difference between the samples with the three different crosslinking degrees. Cell adhesion to PS after 48 h was confirmed by light microscopy.

Cell Adhesion and Spreading on an Intrinsically Anti-Adhesive PEG Biomaterial 405

Fig. 6. Apoptotic level of fibroblasts cultured on PEG-based samples. The induction of apoptosis in L929 cells after 48 h cultivation time on smooth PEG samples was quantified with a caspase-3/-7 assay. The samples had before been extracted in water for 24 h. The test

was performed in triplicate with substrates of three different crosslinking densities.

images of fibroblasts adhering to the micropatterned surface of the PEG hydrogels.

variations of geometry and stiffness of the hydrogels on the enabled cell adhesion.

denoted soft (~90 kPa), intermediate (~350 kPa) and stiff (~1 MPa).

While no cell adhesion was observed on the smooth surface of the PEG-based hydrogels, we have discovered that cells do adhere to bulk PEG hydrogels when they are topographically patterned (Lensen et al., 2008; Schulte et al., 2009). **Figure 7** depicts some representative

In order to explain this observation we have considered that proteins may adsorb to the physically patterned gels and/or that the cells themselves 'feel' the physical pattern and respond on the level of cytoskeletal adaptations. In two follow-up studies we investigated those two (biochemical and biophysical) arguments in more detail. Thus, we examined the possibility of protein adsorption to the gels and we explored the effect of systematic

The first investigation concerned the systematic variation of the deformability of the topographic structures that the cells are assumed to perceive. Although it is unlikely that the cells are able to really deform the micrometer-sized bars, we did envisage that the deformability of the surface structures to depend on the groove width, on the aspect ratio of the bars, and on the inherent mechanical properties of the gels. Thus we prepared line patterns with different groove width (5, 10, 25 and 50 µm) and depth (5, 10 and 15 µm). Also we prepared hydrogel formulations resulting in gels with three different stiffnesses,

**2.4 Fibroblast culture on micropatterned PEG hydrogel substrates** 

Fig. 5. Microscopic investigation of cytocompatibility of PEG-based hydrogels. L929 fibroblasts were cultured on smooth, bulk PEG samples that were fabricated with different percentages (w/v) of photoinitiator (PI) and crosslinking agent (CL); resulting in different mechanical properties. Cell morphology was monitored by light microscopy at different time points (24 h and 48 h) after initial cell seeding. Electron microscopy images (FESEM) of cells which were fixed and dried after 48 h are shown in the bottom row.

Fig. 5. Microscopic investigation of cytocompatibility of PEG-based hydrogels. L929 fibroblasts were cultured on smooth, bulk PEG samples that were fabricated with different percentages (w/v) of photoinitiator (PI) and crosslinking agent (CL); resulting in different mechanical properties. Cell morphology was monitored by light microscopy at different time points (24 h and 48 h) after initial cell seeding. Electron microscopy images (FESEM) of

cells which were fixed and dried after 48 h are shown in the bottom row.

Fig. 6. Apoptotic level of fibroblasts cultured on PEG-based samples. The induction of apoptosis in L929 cells after 48 h cultivation time on smooth PEG samples was quantified with a caspase-3/-7 assay. The samples had before been extracted in water for 24 h. The test was performed in triplicate with substrates of three different crosslinking densities.

#### **2.4 Fibroblast culture on micropatterned PEG hydrogel substrates**

While no cell adhesion was observed on the smooth surface of the PEG-based hydrogels, we have discovered that cells do adhere to bulk PEG hydrogels when they are topographically patterned (Lensen et al., 2008; Schulte et al., 2009). **Figure 7** depicts some representative images of fibroblasts adhering to the micropatterned surface of the PEG hydrogels.

In order to explain this observation we have considered that proteins may adsorb to the physically patterned gels and/or that the cells themselves 'feel' the physical pattern and respond on the level of cytoskeletal adaptations. In two follow-up studies we investigated those two (biochemical and biophysical) arguments in more detail. Thus, we examined the possibility of protein adsorption to the gels and we explored the effect of systematic variations of geometry and stiffness of the hydrogels on the enabled cell adhesion.

The first investigation concerned the systematic variation of the deformability of the topographic structures that the cells are assumed to perceive. Although it is unlikely that the cells are able to really deform the micrometer-sized bars, we did envisage that the deformability of the surface structures to depend on the groove width, on the aspect ratio of the bars, and on the inherent mechanical properties of the gels. Thus we prepared line patterns with different groove width (5, 10, 25 and 50 µm) and depth (5, 10 and 15 µm). Also we prepared hydrogel formulations resulting in gels with three different stiffnesses, denoted soft (~90 kPa), intermediate (~350 kPa) and stiff (~1 MPa).

Cell Adhesion and Spreading on an Intrinsically Anti-Adhesive PEG Biomaterial 407

The cells were found to adhere inside the grooves and form adhesion contacts with the side walls as well as to the bottom of the shallower grooves. They were able to adhere to the narrower (5 µm wide) grooves as well, but had to undergo tremendous shape adaptations, including deformation of the cell nucleus, as observed from fluorescence microscopy using selective staining agents for the actin cytoskeleton and for the nucleus (**Figure 9**, right image). The cells apparently like to snug into well-fitting grooves in a more compliant hydrogel material. No visual deformation of the hydrogels was found; the cells rather adapted their shape to fit into too narrow grooves (i.e. 5 µm wide; Schulte et al., 2010). Second, we investigated whether proteins could adsorb non-specifically to the gels and if so, if they would adsorb differently on the topographic structures, e.g. preferentially on the walls of the grooves, or on the convex (outer) or concave (inner) corners, since the eventually adherent cells were found to be located inside of the grooves and aligned along the ridges. We incubated the patterned hydrogel samples in protein solutions of three selected extracellular matrix (ECM) proteins, i.e. Fibronectin (FN), Vitronectin (VN) and bovine serum albumin (BSA). The former two are cell adhesion mediating proteins, while BSA does not facilitate cell adhesion. BSA is the most abundant protein in serum, and besides its abundance it is also a small protein, which diffuses fast and reaches the surface

With help of immunological staining using fluorescently labeled antibodies we could demonstrate that from pure protein solutions all three proteins were able to adsorb to the PEG surfaces to a certain extent (Schulte et al., 2011). The fluorescence was homogeneously distributed over the surface; there was no detectable difference between for example the vertical walls or the horizontal planes between the grooves. Finally, it seemed that BSA was able to diffuse into the PEG hydrogels, since the fluorescence was not restricted to the

Fig. 9. Elongated cell morphology on topographically patterned PEG hydrogels with 10 or 5 µm wide grooves. Reprinted with permission from: Schulte et al. *Biomacromolecules*, *11,* 3375-

Notwithstanding the ability of all three proteins to adsorb to the PEG hydrogel, only VN was observed to adsorb in a detectable amount under competitive conditions, i.e. from a mixture of VN and FN and when serum, a complex mixture of proteins, was supplemented (**Table 1**; Schulte et al., 2011). This result was rather unexpected, since BSA is usually

faster than the larger proteins FN and VN and finally the cells.

surface (Schulte et al., 2011).

83. Copyright 2010 American Chemical Society.

Fig. 7. Reprinted with permission from: Schulte et al., *Biomacromolecules*, *10,* 2795–2801. Copyright 2009 American Chemical Society.

We have examined the effect of geometric parameters and of mechanical properties of the hydrogels and found that cells prefer to bind inside of grooves that are of comparable size as their cell body, i.e. 10 µm wide and notably when those 10 µm wide grooves were shallow (5 µm deep). The effect of stiffness variations was only evident in combination with topography, and increased cell adhesion and spreading was observed on the softer gels (**Figure 8**; Schulte et al., 2010).

Fig. 8. Cell adhesion (a) and spreading (b) on topographically patterned PEG hydrogels with varied pattern geometries and stiffness. Reprinted with permission from: Schulte et al. *Biomacromolecules*, *11,* 3375-83. Copyright 2010 American Chemical Society.

Fig. 7. Reprinted with permission from: Schulte et al., *Biomacromolecules*, *10,* 2795–2801.

We have examined the effect of geometric parameters and of mechanical properties of the hydrogels and found that cells prefer to bind inside of grooves that are of comparable size as their cell body, i.e. 10 µm wide and notably when those 10 µm wide grooves were shallow (5 µm deep). The effect of stiffness variations was only evident in combination with topography, and increased cell adhesion and spreading was observed on the softer gels

Fig. 8. Cell adhesion (a) and spreading (b) on topographically patterned PEG hydrogels with varied pattern geometries and stiffness. Reprinted with permission from: Schulte et al.

*Biomacromolecules*, *11,* 3375-83. Copyright 2010 American Chemical Society.

Copyright 2009 American Chemical Society.

(**Figure 8**; Schulte et al., 2010).

The cells were found to adhere inside the grooves and form adhesion contacts with the side walls as well as to the bottom of the shallower grooves. They were able to adhere to the narrower (5 µm wide) grooves as well, but had to undergo tremendous shape adaptations, including deformation of the cell nucleus, as observed from fluorescence microscopy using selective staining agents for the actin cytoskeleton and for the nucleus (**Figure 9**, right image). The cells apparently like to snug into well-fitting grooves in a more compliant hydrogel material. No visual deformation of the hydrogels was found; the cells rather adapted their shape to fit into too narrow grooves (i.e. 5 µm wide; Schulte et al., 2010).

Second, we investigated whether proteins could adsorb non-specifically to the gels and if so, if they would adsorb differently on the topographic structures, e.g. preferentially on the walls of the grooves, or on the convex (outer) or concave (inner) corners, since the eventually adherent cells were found to be located inside of the grooves and aligned along the ridges. We incubated the patterned hydrogel samples in protein solutions of three selected extracellular matrix (ECM) proteins, i.e. Fibronectin (FN), Vitronectin (VN) and bovine serum albumin (BSA). The former two are cell adhesion mediating proteins, while BSA does not facilitate cell adhesion. BSA is the most abundant protein in serum, and besides its abundance it is also a small protein, which diffuses fast and reaches the surface faster than the larger proteins FN and VN and finally the cells.

With help of immunological staining using fluorescently labeled antibodies we could demonstrate that from pure protein solutions all three proteins were able to adsorb to the PEG surfaces to a certain extent (Schulte et al., 2011). The fluorescence was homogeneously distributed over the surface; there was no detectable difference between for example the vertical walls or the horizontal planes between the grooves. Finally, it seemed that BSA was able to diffuse into the PEG hydrogels, since the fluorescence was not restricted to the surface (Schulte et al., 2011).

Fig. 9. Elongated cell morphology on topographically patterned PEG hydrogels with 10 or 5 µm wide grooves. Reprinted with permission from: Schulte et al. *Biomacromolecules*, *11,* 3375- 83. Copyright 2010 American Chemical Society.

Notwithstanding the ability of all three proteins to adsorb to the PEG hydrogel, only VN was observed to adsorb in a detectable amount under competitive conditions, i.e. from a mixture of VN and FN and when serum, a complex mixture of proteins, was supplemented (**Table 1**; Schulte et al., 2011). This result was rather unexpected, since BSA is usually

Cell Adhesion and Spreading on an Intrinsically Anti-Adhesive PEG Biomaterial 409

We analyzed the number of adherent cells and found an increased number of cells on the VN-incubated hydrogels. This effect was also found for the unpatterned, smooth hydrogels; after VN-incubation a very small but significant number of cells were able to adhere to the PEG surface. In **Figure 10** these results are depicted; comparing smooth and patterned

It can be seen that compared to the effect of topography alone, the VN-incubation alone was less effective in enabling cell adhesion. Remarkably, the effect of VN on cell adhesion was only evident at early time points; after 24 hours the enabling effect was completely lost. Finally, a striking synergistic effect was observed from the combination of VN-incubation and topography; the number of adherent and spread cells was larger than the sum of the individual contributions (Schulte et al., 2011). Taking into account the apparent difference in the effect of topography and VN with time, we tentatively conclude that the cell adhesion protein VN facilitates the initial cell adhesion, while the adhesion-enabling effect of surface topography becomes dominant at longer times and is necessary for the development of

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

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

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

hydrogels with and without VN-incubation.

durable and stable adhesion complexes.

achieved by varying the crosslinking density.

adhesion to the intrinsically anti-adhesive PEG biomaterial.

**3. Conclusion** 

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 much more studied than VN.


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

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

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. We analyzed the number of adherent cells and found an increased number of cells on the VN-incubated hydrogels. This effect was also found for the unpatterned, smooth hydrogels; after VN-incubation a very small but significant number of cells were able to adhere to the PEG surface. In **Figure 10** these results are depicted; comparing smooth and patterned hydrogels with and without VN-incubation.

It can be seen that compared to the effect of topography alone, the VN-incubation alone was less effective in enabling cell adhesion. Remarkably, the effect of VN on cell adhesion was only evident at early time points; after 24 hours the enabling effect was completely lost. Finally, a striking synergistic effect was observed from the combination of VN-incubation and topography; the number of adherent and spread cells was larger than the sum of the individual contributions (Schulte et al., 2011). Taking into account the apparent difference in the effect of topography and VN with time, we tentatively conclude that the cell adhesion protein VN facilitates the initial cell adhesion, while the adhesion-enabling effect of surface topography becomes dominant at longer times and is necessary for the development of durable and stable adhesion complexes.
