Silky gels for cells : Self-assembling protein-based polymers for use in tissue engineering

Tissue engineering is a relatively new, but actively developing field of biomedical science. It aims at organ or tissue regeneration by use of scaffolds, which are usually seeded with cells prior to implantation, and stimulated by bioactive cues or growth factors. It is a promising and valuable alternative to the use of transplants, for which the demand is greater than the supply, and for which application is connected with high risk of rejection and infection due to immunosuppressant medication. One of the main challenges of tissue engineering, that we tried to address in this thesis, is the design of biocompatible and functional biomaterials that could serve as cell scaffold. We investigated, if protein-based polymers, more specifically, if the <em>de novo</em> designed, <strong>C2SH48C2</strong> copolymer, which self-assembles into fibers upon a pH-trigger, is a suitable material for cell scaffolds. In <strong>Chapter 2</strong> we described the design and production, by means of recombinant DNA technology, of <strong>C2SH48C2</strong>. The protein was efficiently secreted by <em>Pichia pastoris </em>at high yields of g/l levels and we proposed an effective purification method. We showed that fibers and gels form by self-assembly upon pH adjustment, and that rheological properties of the obtained hydrogels depend on the total protein concentration. In view of potential biomedical applications, erosion studies were performed, which indicated that the gels exhibited long term stability in conditions mimicking those in body fluid. The biocompatibility of the gel scaffolds was demonstrated in a 2D cell culture study. However, despite the cell viability, a low proliferation rate was observed. To improve cell performance in contact with <strong>C2SH48C2 </strong>hydrogels (<strong>Chapter 3</strong>) we incorporated active domains in the <strong>C2SH48C2</strong> protein by recombinant functionalization. We described the synthesis of two protein variants: (1) <strong>BRGDC2SH48C2</strong>, N-terminally enriched in integrin-binding domains (RGD) and (2) <strong>BKRSRC2SH48C2</strong>, N-terminally enriched in heparin binding domains (KRSR). We showed precise control over the amount of active domains in the final gels, by simply mixing the variants of the proteins in the desired molar ratio before inducing gelation. A 23-day cell culture study, performed using MG-63 cells, revealed that the presence of RGD and KRSR domains positively influenced cell attachment, spreading and activity. A synergistic effect was observed, i.e. scaffolds containing both bioactive domains yielded fully confluent layers of cells at an earlier stage during cell culture than the other gels. We concluded that cell behavior can be controlled by tuning the content of functional domains. In <strong>Chapter 4</strong>, we tested the suitability of the <strong>C2SH48C2</strong> protein, enriched in RGD domains, for cell encapsulation, as the conditions of 3D cell culturing are more similar to the environment of cells in the body. We independently varied gel stiffness (by means of protein concentration) and functional motif (RGD) density, and analyzed the influence of these parameters on the cellular response. The viability and proliferation of MG-63 cells, encapsulated in the gels at different protein concentrations and RGD densities, was investigated with a cell activity assay, and by quantitative analysis of confocal pictures of nuclei (DAPI stain) and F-actin (phalloidin). We showed that optimal cell behavior is obtained in the presence of RGD domains and at low protein concentrations. The results indicated that RGD functionality is not the sole requirement; the gel matrix needs to exhibit the right mechanical properties and architecture to allow for cell growth, cytoplasmic extension and migration. Finally, in <strong>Chapter 5</strong>, we showed that active domains (here KRSR) can serve multiple functions in the material. We demonstrated the cross-linking ability of KRSR domains in the presence of heparin, leading to structural and mechanical changes in the scaffolds. In dilute systems (0.1 % (w/v)), heparin increases the rate of fiber growth, and induces fiber bundling. At higher protein concentrations, leading to the hydrogel formation (2 % (w/v)), the gelation rate and final storage modulus can be tuned by the amount of heparin and KRSR domain density. We concluded that with this approach, the material properties of <strong>C2SH48C2</strong> protein gels can be effectively and simply controlled in a straightforward and biocompatible way. In <strong>Chapter 6</strong> we described the main requirements for biomaterials and discussed to what extent they are fulfilled by protein-based polymers, and in particular, by the presented <strong>C2SH48C2</strong> protein. The main advantages over alternative materials, and the challenges that need to be addressed before application in tissue engineering becomes a reality, were discussed. We ended with suggestions to improve the properties of <strong>C2SH48C2</strong> protein for use as a biomaterial, especially its biodegradability, and its structural and mechanical properties.