**1.2 Biomedical applications of PEG- or PEO-based hydrogels**

Some of the earliest work on the use of PEG and PEO as hydrophilic biomaterials showed that PEO adsorption onto glass surfaces prevented protein adsorption (Merrill et al., 1982). Several subsequent studies confirmed that PEO, or its low molecular weight (Mw<10 kDa) equivalent, PEG, were showing the most effective protein-repellent properties (Harris, 1992). PEG-modified surfaces are non-permissive to protein adsorption, bacterial adhesion and eukaryotic cell adhesion (Zhang et al., 1998; Desai et al., 1992; Drumheller et al., 1995; Krsko et al., 2009.

Based on these properties, PEG hydrogels are one of the most widely studied and used materials for a variety of biomedical applications such as tissue engineering, coating of implants, biosensors, and drug delivery systems (Langer & Peppas, 2003; Langer & Tirell, 2004; Krsko & Libera, 2005; Tessmar & Gopferich, 2007; Veronese & Mero, 2008; Harris & Zalipsky, 1997). PEG substrates have also been used to generate patterns of proteins or cells using for example the technique of microcontact printing (Whitesides et al., 2001; Mrksich & Whitesides, 1996; Mrksich et al., 1997). PEG hydrogels are approved by the US Food and Drug Administration (FDA) for oral and topical application; they are little immunogenic and non-toxic at molecular weights above 400 Da. Since PEG itself is not degradable by simple hydrolysis and undergoes only limited metabolism in the body, the whole polymer chains are eliminated through the kidneys or eventually through the liver (Mw < 30 kDa) (Harris, 1992; Knauf et al., 1988).

Many groups have investigated surface coverings of PEG or PEO in order to try to elucidate why PEG has such remarkably effective properties and different theories have been proposed (Jeon et al., 1991; Prime & Whitesides, 1993). First, there are generally only weak attractive interactions between the PEG-coatings and a wide range of proteins, as protein adsorption is generally known to be more pronounced on hydrophobic surfaces in comparison to hydrophilic ones (Morra, 2000). Furthermore, as the interaction between water and PEG via hydrogen bonds is more favorable and surpasses possible attractive interactions of proteins with the surfaces, a repulsion force is created. Therefore the hydration of the layer, i.e. the binding of interfacial water is of high relevance for the exclusion of other molecules coming near the polymer surface (Harris, 1992; Harder et al., 1998). Additionally, molecules approaching the rather flexible, loosely crosslinked PEG hydrogel from the surrounding medium initiate the compression of the extended PEG molecules inducing a steric repulsion effect (Jeon et al., 1991; Morra, 2000). More specifically, a loosely crosslinked gel has relatively long segments between the crosslinks, which can take a relatively large number of conformations. The number of segment conformations would be substantially restricted by the binding of a protein molecule to the gel surface. This

Cell Adhesion and Spreading on an Intrinsically Anti-Adhesive PEG Biomaterial 401

To ensure a complete reaction of the acrylate end-groups which could otherwise undergo undesired reactions with the biological system, the curing kinetics of the system were monitored. It was confirmed that after 10 min more than 90 % of the C-C double bonds of the acrylate end-groups had been consumed. After 60 min only 2.3 % of unreacted endgroups were left. Based on these observations it was decided to apply 60 min of UVirradiation to the samples in order to achieve virtually complete crosslinking. Bulk PEGbased substrates were fabricated by casting the prepolymer mixture against a smooth silicon

Fig. 3. Young's modulus (MPa) of bulk PEG-based hydrogel samples in dry and swollen state; gels were fabricated from three precursor mixtures with different percentages (w/v) of photoinitiator (PI) and crosslinking agent (CL). Reprinted with permission from: Schulte

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

Fig. 2. Fabrication of bulk PEG-based hydrogels by means of UV-curing.

surface.

would lead to a relatively large unfavorable entropic change, making the process of protein adsorption very unfavorable for thermodynamic reasons. Additionally, the high mobility of PEG chains allows little time for proteins to form durable attachments.

Many techniques have been developed to create PEG or PEO-bearing surfaces, e.g. exploiting physical adsorption, chemical coupling, and graft polymerization (Harris, 1992; Harris & Zalipsky, 1997; Prime & Whitesides, 1993; Fujimoto et al., 1993; Prime & Whitesides, 1991). Whitesides and co-workers have studied covalent coatings of oligo(ethylene glycol)s, so-called self-assembled monolayers (SAMs) and found that the resistance to protein adsorption increased with the chain length of the oligomers (Prime & Whitesides, 1991 and 1993). Furthermore, it has been demonstrated that the adhesion resistance of PEG increases with chain packing density (Sofia et al., 1998; Malmsten et al., 1998).

In recent years the versatility of star-shaped PEG molecules has been recognized, as they present a high number of end-groups per molecule allowing interconnectivity and functionalization (Groll et al., 2005a & 2005b; Lutolf et al., 2003). Some star polymers have been shown to achieve a high surface coverage and localization of the end-groups near the top of the star polymer (Irvine et al., 1996). Therefore, star-shaped PEG molecules are an interesting and promising alternative to linear PEG.

#### **1.3 PEG-based hydrogels formed by UV-curing: patternable biomaterials**

We have been using PEG hydrogels that are prepared by UV-based radical crosslinking of six-armed star-shaped macromonomers via acrylate (Acr) end-groups. The polymer backbone consists of a statistical copolymer of 85 % ethylene oxide and 15 % propylene oxide (P(EO-stat-PO)) and each star molecule bears 6 reactive Acr end-groups. The formal notation of the precursor polymer would thus be Acr sP(EO stat PO). Nevertheless, in the following the resulting, crosslinked network will be denoted PEG-based (hydro)gel, even though the arms of the precursors do not consist of pure PEG, but contain a fraction (15%) of propylene glycol units in the copolymer. These PO-units give the prepolymer its unique and very useful property of being a liquid at room temperature, before crosslinking. The crosslinking reaction was initiated by a UV-based radical reaction with benzoin methyl ether as photoinitiator (PI) and an additional crosslinker (CL) (pentaerythritol triacrylate). Further experimental details concerning the synthesis and the curing conditions can be found in our recent publications (Lensen et al., 2007; Diez et al., 2009).

The hydrogel substrates were applied as free-standing bulk gels for 2D cell culture studies. Due to the fact that the prepolymer Acr-sP(EO-stat-PO) is liquid before crosslinking, the precursor mixture can be molded in any shape, which has enabled us to imprint desired micro- and nanometer topographic patterns into the hydrogel surface (Lensen et al., 2007; Diez et al., 2009). In the following, the properties of this hydrogel system in view of its use in biomedical applications will be evaluated, e.g. the cytotoxicity and cytocompatibility will be assessed, and the cell behavior on the surface of the hydrogels will be demonstrated. Finally, the remarkable effect of surface topography and substrate elasticity on protein adsorption, cell adhesion and cell spreading will be discussed.

#### **2. Fabrication and properties of PEG-based substrates**

#### **2.1 Synthesis of PEG-based hydrogels from Acr-sP(EO-stat-PO) macromonomers**

Hydrogels fabricated for the application in cell culture studies were crosslinked from AcrsP(EO-stat-PO) prepolymers. UV-irradiation was used to initiate radical polymerization of the macromonomer mixture with added photoinitiator (PI) and crosslinking agent (CL) (**Figure 2**).

would lead to a relatively large unfavorable entropic change, making the process of protein adsorption very unfavorable for thermodynamic reasons. Additionally, the high mobility of

Many techniques have been developed to create PEG or PEO-bearing surfaces, e.g. exploiting physical adsorption, chemical coupling, and graft polymerization (Harris, 1992; Harris & Zalipsky, 1997; Prime & Whitesides, 1993; Fujimoto et al., 1993; Prime & Whitesides, 1991). Whitesides and co-workers have studied covalent coatings of oligo(ethylene glycol)s, so-called self-assembled monolayers (SAMs) and found that the resistance to protein adsorption increased with the chain length of the oligomers (Prime & Whitesides, 1991 and 1993). Furthermore, it has been demonstrated that the adhesion resistance of PEG increases with

In recent years the versatility of star-shaped PEG molecules has been recognized, as they present a high number of end-groups per molecule allowing interconnectivity and functionalization (Groll et al., 2005a & 2005b; Lutolf et al., 2003). Some star polymers have been shown to achieve a high surface coverage and localization of the end-groups near the top of the star polymer (Irvine et al., 1996). Therefore, star-shaped PEG molecules are an

We have been using PEG hydrogels that are prepared by UV-based radical crosslinking of six-armed star-shaped macromonomers via acrylate (Acr) end-groups. The polymer backbone consists of a statistical copolymer of 85 % ethylene oxide and 15 % propylene oxide (P(EO-stat-PO)) and each star molecule bears 6 reactive Acr end-groups. The formal notation of the precursor polymer would thus be Acr sP(EO stat PO). Nevertheless, in the following the resulting, crosslinked network will be denoted PEG-based (hydro)gel, even though the arms of the precursors do not consist of pure PEG, but contain a fraction (15%) of propylene glycol units in the copolymer. These PO-units give the prepolymer its unique and very useful property of being a liquid at room temperature, before crosslinking. The crosslinking reaction was initiated by a UV-based radical reaction with benzoin methyl ether as photoinitiator (PI) and an additional crosslinker (CL) (pentaerythritol triacrylate). Further experimental details concerning the synthesis and the curing conditions can be found in our

The hydrogel substrates were applied as free-standing bulk gels for 2D cell culture studies. Due to the fact that the prepolymer Acr-sP(EO-stat-PO) is liquid before crosslinking, the precursor mixture can be molded in any shape, which has enabled us to imprint desired micro- and nanometer topographic patterns into the hydrogel surface (Lensen et al., 2007; Diez et al., 2009). In the following, the properties of this hydrogel system in view of its use in biomedical applications will be evaluated, e.g. the cytotoxicity and cytocompatibility will be assessed, and the cell behavior on the surface of the hydrogels will be demonstrated. Finally, the remarkable effect of surface topography and substrate elasticity on protein adsorption,

**2.1 Synthesis of PEG-based hydrogels from Acr-sP(EO-stat-PO) macromonomers**  Hydrogels fabricated for the application in cell culture studies were crosslinked from AcrsP(EO-stat-PO) prepolymers. UV-irradiation was used to initiate radical polymerization of the macromonomer mixture with added photoinitiator (PI) and crosslinking agent (CL) (**Figure 2**).

**1.3 PEG-based hydrogels formed by UV-curing: patternable biomaterials** 

PEG chains allows little time for proteins to form durable attachments.

chain packing density (Sofia et al., 1998; Malmsten et al., 1998).

interesting and promising alternative to linear PEG.

recent publications (Lensen et al., 2007; Diez et al., 2009).

cell adhesion and cell spreading will be discussed.

**2. Fabrication and properties of PEG-based substrates** 

Fig. 2. Fabrication of bulk PEG-based hydrogels by means of UV-curing.

To ensure a complete reaction of the acrylate end-groups which could otherwise undergo undesired reactions with the biological system, the curing kinetics of the system were monitored. It was confirmed that after 10 min more than 90 % of the C-C double bonds of the acrylate end-groups had been consumed. After 60 min only 2.3 % of unreacted endgroups were left. Based on these observations it was decided to apply 60 min of UVirradiation to the samples in order to achieve virtually complete crosslinking. Bulk PEGbased substrates were fabricated by casting the prepolymer mixture against a smooth silicon surface.

Fig. 3. Young's modulus (MPa) of bulk PEG-based hydrogel samples in dry and swollen state; gels were fabricated from three precursor mixtures with different percentages (w/v) of photoinitiator (PI) and crosslinking agent (CL). Reprinted with permission from: Schulte et al. *Biomacromolecules*, *11,* 3375-83. Copyright 2010 American Chemical Society.

Cell Adhesion and Spreading on an Intrinsically Anti-Adhesive PEG Biomaterial 403

The percentage of dead cells after 24 h in the indirect cell test with medium that had been conditioned with PEG samples gave values between 7 % and 16 % depending on the sample composition (variations in the amount of PI and CL) and the solvent used for previous extraction of the samples. No significant difference in cytotoxicity could be observed comparing soft, intermediate and stiff samples. Looking at the impact of sample extraction medium, substrates which had been incubated in water for 24 h prior to the test showed the lowest cytotoxic effect. Compared with results gained by extraction with organic solvents (acetone) or without any treatment, the cytotoxicity level detected for the water washed samples was significantly lower (p<0.05 and p< 0.01, respectively). As a consequence of these test results PEG samples were stored in sterile water for at least 24 h after the

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

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

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

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

crosslinking process prior to in vitro application.

to PS after 48 h was confirmed by light microscopy.

microscopy (FESEM).

the surface.

in **Figure 6**.

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

The UV-photopolymerization via the acrylate end-groups on the sP(EO-stat-PO) arms does not only allow topographic patterning of the hydrogel's surface, but also enables tuning of the crosslinking density, hence stiffness. Thus, varying the amount of added photoinitiator (PI) and crosslinker (CL) represents a practical approach to controlling the mechanical properties of PEG-based hydrogels (**Figure 3**). This is of high relevance for biomedical applications, as it is well known that cells feel and respond to the stiffness of the underlying substrate (Discher et al., 2005; Engler et al., 2006; Yeung et al., 2005). PEG-based hydrogels with distinctly different mechanical properties were fabricated; the resulting hydrogels from 3 different formulations are denoted as soft, intermediate and stiff (Schulte et al., 2010; Diez et al., 2011).

The stiffness in the swollen state, which is obviously the most relevant for cell culture, was shown to be approximately half of that measured in the dry state, ranging from ~100 kPa for the softest to 1 MPa for the stiffest, thus covering one order of magnitude in elastic modulus (Figure 3).

#### **2.2 Cytotoxicity assessment of PEG-based substrates**

As the material has not been used in this exact composition before, cytotoxicity tests were conducted to prove that possible traces of unreacted acrylate end-groups, photoinitiator or crosslinker did not interfere with cell viability. The impact of the material on the viability of L929 fibroblasts was tested with an indirect cell test. Cell membrane integrity as an important indicator for cell viability was tested with a commercially available enzymatic assay. Values shown in **Figure 4** were derived by comparison with a control sample of mortalized cells (incubation with DMSO), which was set to 100 % cytotoxicity. It should be noted that no direct test was possible as PEG is known to be anti-adhesive and the majority of the seeded cells would not stay attached to the PEG-substrate and could therefore not be used for quantitative studies.

Fig. 4. Cytotoxicity of bulk PEG samples; shown are the results of an indirect cytotoxicity test with L929 cultured for 24 h in PEG conditioned (72 h) medium. The PEG samples had before been extracted in water, acetone or methanol for 24 h. The test was performed in triplicate with substrates of three different crosslinking densities. Statistical significance indicated by \*\*: p<0.01; \*: p<0.05.

The UV-photopolymerization via the acrylate end-groups on the sP(EO-stat-PO) arms does not only allow topographic patterning of the hydrogel's surface, but also enables tuning of the crosslinking density, hence stiffness. Thus, varying the amount of added photoinitiator (PI) and crosslinker (CL) represents a practical approach to controlling the mechanical properties of PEG-based hydrogels (**Figure 3**). This is of high relevance for biomedical applications, as it is well known that cells feel and respond to the stiffness of the underlying substrate (Discher et al., 2005; Engler et al., 2006; Yeung et al., 2005). PEG-based hydrogels with distinctly different mechanical properties were fabricated; the resulting hydrogels from 3 different formulations

The stiffness in the swollen state, which is obviously the most relevant for cell culture, was shown to be approximately half of that measured in the dry state, ranging from ~100 kPa for the softest to 1 MPa for the stiffest, thus covering one order of magnitude in elastic modulus

As the material has not been used in this exact composition before, cytotoxicity tests were conducted to prove that possible traces of unreacted acrylate end-groups, photoinitiator or crosslinker did not interfere with cell viability. The impact of the material on the viability of L929 fibroblasts was tested with an indirect cell test. Cell membrane integrity as an important indicator for cell viability was tested with a commercially available enzymatic assay. Values shown in **Figure 4** were derived by comparison with a control sample of mortalized cells (incubation with DMSO), which was set to 100 % cytotoxicity. It should be noted that no direct test was possible as PEG is known to be anti-adhesive and the majority of the seeded cells would not stay attached to the PEG-substrate and could therefore not be

Fig. 4. Cytotoxicity of bulk PEG samples; shown are the results of an indirect cytotoxicity test with L929 cultured for 24 h in PEG conditioned (72 h) medium. The PEG samples had before been extracted in water, acetone or methanol for 24 h. The test was performed in triplicate with substrates of three different crosslinking densities. Statistical significance

are denoted as soft, intermediate and stiff (Schulte et al., 2010; Diez et al., 2011).

**2.2 Cytotoxicity assessment of PEG-based substrates** 

(Figure 3).

used for quantitative studies.

indicated by \*\*: p<0.01; \*: p<0.05.

The percentage of dead cells after 24 h in the indirect cell test with medium that had been conditioned with PEG samples gave values between 7 % and 16 % depending on the sample composition (variations in the amount of PI and CL) and the solvent used for previous extraction of the samples. No significant difference in cytotoxicity could be observed comparing soft, intermediate and stiff samples. Looking at the impact of sample extraction medium, substrates which had been incubated in water for 24 h prior to the test showed the lowest cytotoxic effect. Compared with results gained by extraction with organic solvents (acetone) or without any treatment, the cytotoxicity level detected for the water washed samples was significantly lower (p<0.05 and p< 0.01, respectively). As a consequence of these test results PEG samples were stored in sterile water for at least 24 h after the crosslinking process prior to in vitro application.
