Biofunktionalisierung von Biomaterialoberflächen mit poly-LacNAc und den Galektinen-1, -3 und -8
- Biofunctionalization of biomaterial surfaces with poly-LacNAc and the galectins-1, -3 and -8
Rech, Claudia; Elling, Lothar (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)
Aachen : Publikationsserver der RWTH Aachen University (2015)
Dissertation / PhD Thesis
Aachen, Techn. Hochsch., Diss., 2014
The aim of this work was to build up a modular, artificial extracellular matrix (ECM) with poly-LacNAc, the human galectins-1, -3 and -8 and ECM-glycoproteins on a potential biomaterial surface. First, the synthesis of poly-LacNAc was characterized, optimized and up-scaled. This included the optimization of the single steps from enzyme expression and purification over synthesis till product isolation. The characterization of the one-pot-synthesis of the poly-LacNAc mixture was prioritized. The ratio of the substrates and the enzymes as well as their amount was optimized. During characterization of one-pot-synthesis with different enzyme ratios, I observed an unexpected product distribution which could be explained by kinetic data with longer substrates: The enzyme beta3GlcNAcT has higher activity to the tetra-saccharide. The synthesis based on acceptor substrates of different length showed a limitation of chain length from nona-saccharid onwards. In semi-preparative synthesis, poly-LacNAc structures till hexadeca-saccharides were detected and poly-LacNAc till dodeca-saccharides were isolated for the first time. Second, the human galectins-1, -3 and -8 were characterized with respect to their use for building up a modular, biomimetic ECM. Furthermore, common proteinchemical questions like stability and influence of His-Tag were discussed. It was possible to cross-link the ECM-glycoproteins laminin and fibronectin with all three galectins (His6Gal-1C2S, His6Gal-3 und His6Gal-8) on immobilized poly-LacNAc structures. His6Gal-1C2S binds already on the di-saccharid LacNAc on hydrogels. In contrast, the galectins-3 and -8 as well as the model galectin CGL2 need longer chain length for optimal binding on poly-LacNAc on hydrogels. Consequently, the poly-LacNAc mixture is the optimum for the first construction without any specific epitopes. Third, a new screening system with hydrogel in the microtiterplate was developed. Consequently, the galectins could be characterized on the final biomaterial surface. Finally, the biomimetic surface was tested as proof-of-principle with fibroblasts. They showed very good adhesion and proliferation on the human galectins (bound on poly-LacNAc) and on the surface poly-LacNAc-galectin-fibronectin. Furthermore, the biomimetic ECM (with model galectin CGL2) is more adhesive in comparison to the standard surface TCPS (tissue culture plates poly styrene) and to immobilized fibronectin. I hypothesized that the already established ECM leads to an increase of adhesion and proliferation of fibroblasts because no build-up of ECM is necessary. Additionally, the biomimetic ECM is more flexible than immobilized fibronectin and can be rearranged by adherent cells.