**1.1 Application of hydrogels in biomaterial science**

Amongst the different classes of materials which find use in the field of medicine and biology, hydrophilic polymers have demonstrated great potential. Networks formed from hydrophilic polymer often exhibit a high affinity for water, yet they do not dissolve due to their chemically or physically crosslinked network. Water can penetrate in between the chains of the polymer network, leading to swelling and the formation of a hydrogel (Langer & Peppas, 2003; Peppas et al., 2000; Wichterle & Lim, 1960). Generally such polymer networks can be formed via chemical bonds, ionic interactions, hydrogen bonds, hydrophobic interactions, or physical bonds (Hoffman, 2002; Peppas, 1986). Hydrogels have found numerous applications in drug delivery as well as in tissue engineering where they are used as scaffolds for the cultivation of cells to enable the formation of new tissues (Jen et al. 1996; Krsko & Libera, 2005; Langer & Tirrell, 2004; Peppas et al., 2006). Hydrogels are especially attractive for this purpose as they meet numerous characteristics of the architecture and mechanics of most soft tissues and many are considered biocompatible (Jhon & Andrade, 1973; Saha et al., 2007). Furthermore, concerning the intended purpose of cell encapsulation and delivery, hydrogels support sufficient transport of oxygen, nutrients and wastes (Fedorovich et al., 2007; Lee & Mooney, 2001; Nguyen & West, 2002).

In general, hydrogel matrices can be prepared from a variety of naturally derived proteins and polysaccharides, as well as from synthetic polymers (Peppas et al., 2006). Depending on their origin and composition, natural polymers have specific utilities and properties. Hydrogels from natural sources have for example been fabricated from collagen, hyaluronic acid (HA), fibrin, alginate and agarose (Hoffman, 2002). Collagen, HA and fibrin are

Cell Adhesion and Spreading on an Intrinsically Anti-Adhesive PEG Biomaterial 399

circulating blood, begins with the unspecific adsorption of plasma proteins (Andrade &

Not only with regards to tissue engineering and implant design unspecific protein adsorption is a highly critical process, also different devices in diagnostics (e.g. protein arrays) and biosensors are based on specific receptor-ligand binding, demanding a noninteracting background. Therefore, much effort has been focused on the development of inert, protein resistant materials and coatings (Chapman et al., 2000; Elbert & Hubbell, 1996). Many synthetic hydrophilic polymers, including PAA, PHEMA, PVA, PEG and poly(ethylene oxide) (PEO) have been applied for this purpose (see Figure 1) (Castillo et al.,

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;

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)

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

Hlady, 1986; Harris, 1992; Horbett, 1993).

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

1985).

Krsko et al., 2009.

(Harris, 1992; Knauf et al., 1988).

components which are in vivo present in the extracellular matrix (ECM) of mammalian cells. Since they are derived from natural sources, hydrogels formed from these polymers are inherently cytocompatible and bioactive. They can promote many cellular functions due to a diversity of endogenous factors present. However, scaffolds fabricated from natural sources are rather complex and often ill-defined, making it difficult to determine exactly which signals are promoting the cellular outcome (Cushing & Anseth, 2007). Furthermore they can possess an inherent batch-to-batch variability which can affect sensitive cells in their viability, proliferation, and development (Cushing & Anseth, 2007). Due to these limitations of gels formed from natural polymers, a wide range of synthetic polymers has been found suitable regarding their chemical and physical properties (Hoffman, 2002). The advantages of synthetic gels include their consistent composition and predictable manipulation of properties.

A few examples of synthetic hydrogel building blocks are given in **Figure 1**, including neutral (upper row) and ionic (bottom row) monomers (Peppas et al., 2006).

Fig. 1. Some examples of synthetic hydrogels that are used in biomedical applications. Reproduced with permission from Peppas et al., *Adv. Mater.*, *18*, 1345-60. Copyright 2006 John Wiley and Sons.

Proteins are molecules, which often adsorb unspecifically from solution at biomaterial interfaces, a phenomenon that has been documented in a wealth of publications, e.g. references: (Andrade & Hlady, 1986; Andrade et al., 1992; Wahlgren & Arnebrant, 1991). Almost any material, when exposed to a physiological, protein-containing solution, becomes coated with proteins within seconds. As widely recognized, this adsorption of proteins to synthetic material surfaces is of great importance in the field of biomaterials as it plays a determining role for the subsequent cellular responses. Failure of most implant materials stems from an inability to predict and control the process of protein adsorption and cell interaction, resulting in an inappropriate host response to the material (Castner & Ratner, 2002; Hlady & Buijs, 1996; Tsai et al., 2002). Biomaterial surface-induced thrombosis, for example, one of the major problems in clinical applications of materials in contact with

components which are in vivo present in the extracellular matrix (ECM) of mammalian cells. Since they are derived from natural sources, hydrogels formed from these polymers are inherently cytocompatible and bioactive. They can promote many cellular functions due to a diversity of endogenous factors present. However, scaffolds fabricated from natural sources are rather complex and often ill-defined, making it difficult to determine exactly which signals are promoting the cellular outcome (Cushing & Anseth, 2007). Furthermore they can possess an inherent batch-to-batch variability which can affect sensitive cells in their viability, proliferation, and development (Cushing & Anseth, 2007). Due to these limitations of gels formed from natural polymers, a wide range of synthetic polymers has been found suitable regarding their chemical and physical properties (Hoffman, 2002). The advantages of synthetic gels include their consistent composition and predictable manipulation of

A few examples of synthetic hydrogel building blocks are given in **Figure 1**, including

Fig. 1. Some examples of synthetic hydrogels that are used in biomedical applications. Reproduced with permission from Peppas et al., *Adv. Mater.*, *18*, 1345-60. Copyright 2006

Proteins are molecules, which often adsorb unspecifically from solution at biomaterial interfaces, a phenomenon that has been documented in a wealth of publications, e.g. references: (Andrade & Hlady, 1986; Andrade et al., 1992; Wahlgren & Arnebrant, 1991). Almost any material, when exposed to a physiological, protein-containing solution, becomes coated with proteins within seconds. As widely recognized, this adsorption of proteins to synthetic material surfaces is of great importance in the field of biomaterials as it plays a determining role for the subsequent cellular responses. Failure of most implant materials stems from an inability to predict and control the process of protein adsorption and cell interaction, resulting in an inappropriate host response to the material (Castner & Ratner, 2002; Hlady & Buijs, 1996; Tsai et al., 2002). Biomaterial surface-induced thrombosis, for example, one of the major problems in clinical applications of materials in contact with

neutral (upper row) and ionic (bottom row) monomers (Peppas et al., 2006).

properties.

John Wiley and Sons.

circulating blood, begins with the unspecific adsorption of plasma proteins (Andrade & Hlady, 1986; Harris, 1992; Horbett, 1993).

Not only with regards to tissue engineering and implant design unspecific protein adsorption is a highly critical process, also different devices in diagnostics (e.g. protein arrays) and biosensors are based on specific receptor-ligand binding, demanding a noninteracting background. Therefore, much effort has been focused on the development of inert, protein resistant materials and coatings (Chapman et al., 2000; Elbert & Hubbell, 1996). Many synthetic hydrophilic polymers, including PAA, PHEMA, PVA, PEG and poly(ethylene oxide) (PEO) have been applied for this purpose (see Figure 1) (Castillo et al., 1985).
