Polymers and protein-conjugates for tissue engineering

The number of patients in our society needing replacement of severed, missing or malfunctioning tissue due to diseases, traumata or age, is high and still growing. Burn wounds, bone diseases, diabetes or liver insufficiency are examples of such suffering. Conventional attempts of remedy like drug supplementation, mechanical devices or xenografts are often unsatisfactory and fall short of the goal of enabling normal life. An innovative therapy approach is Human Tissue Engineering, a discipline dedicated to the field of regenerative medicine. To fulfill the promises raised by first successes, significant research efforts have been designated on this area, focusing on various subjects, such as cells, scaffold materials, culture conditions, bioactive molecules, and others. An important milestone on the route for Tissue Engineered products, was the use of �intelligent� materials, such as biomimetic polymers, which may themselves influence cell adhesion and direct cell development (Chapter 1). Biomimetic substances are designed to mimic a biological environment, insomuch eliciting cellular responses that help the material to better perform the intended task. A polymer with favorable characteristics with regards to its use as scaffold material for Tissue Engineering applications is the poly(ethylene glycol)-block-poly(lactic acid) copolymer. The poly(ethylene glycol) block is responsible for suppression of unspecific protein adsorption, whereas the main advantages of the poly(lactic acid) block is its biodegradability. If the free group of the poly(ethylene glycol) block provides a reactive group, the polymer is easily processed into a biomimetic polymer, by covalent attachment of a bioactive molecule, such as bFGF. Diblock co-polymers with different molecular weight, namely MePEG2PLA20, MePEG2PLA40, and NH2PEG2PLA40 were synthesized and characterized with the goal of obtaining substances processable to mechanically stable scaffolds for in vivo or in vitro experiments and first steps were undertaken towards the in vivo evaluation of these polymers (Chapter 4). Although PEG chains largely suppress undesired protein adsorption, the shielding of the surface is not complete. As randomly adsorbed proteins are likely to interfere with the targeted tissue development, knowledge on the extent of shielding is crucial. We, therefore, investigated the amount of adsorbed protein to different polymers by processing them into thin films and investigating their interactions with proteins using quartz crystal microbalance techniques (Chapter 5). Covalent linking of molecules to polymers has the advantage of constricting their operating range to the tethering site, thus diminishing undesired side-effects. Moreover, as diffusion of these molecules is inhibited, prolonged reaction times might be an additional benefit. Unfortunately, tethering might interfere with conformational changes of the molecules structure required for its interaction with the substrate or might even affect the active center of the molecule. Therefore, knowledge of the reaction site is crucial. Identification of the reaction sites was attempted using insulin as model protein. Therefore, polymers were linked to the proteins via small molecular weight tags. After removal of the polymer chains the succinic acid tags still marked the reaction sites and the modified proteins were characterized to identify the exact location of these tags (Chapter 6). Chemical modification of proteins might alter the physico-chemical properties of the molecules. For example, tethering of hydrophilic polymers, like PEG, enhances the water solubility of the drug. But also modification of proteins by attachment of hydrophobic substances, such as fatty acids, might improve the characteristics of these proteins. Nature often uses fatty acids as membrane tethering moieties. Lipidized proteins are also candidates for alternative administration routes, like the oral route, being more stable and having enhanced adsorption properties. Unfortunately, modification of proteins might deteriorate their biological activity. Therefore, prior to use each synthesized conjugate must be tested in a suitable model system. Here, insulin was chosen as model protein. After attachment of fatty acids, its biological activity was tested in an insulin dependent chondrocyte cell culture system (Chapter 7).