Thesis presented December 15, 2021

Abstract:
The extra cellular matrix (ECM) provides mechanical structure to embedded cells and gives biochemical cues to regulate homeostasis and differentiation. The structural molecule fibronectin contains adhesion motifs such as the RGD sequence with high specificity for β1- and β3 -integrins but also binding domains for the glycosaminoglycan (GAG) heparan sulfate (HS). GAGs have a high swelling capacity due to their negative charge but also interact with growth factors to protect them from degradation and thus serve as a reservoir. HS binds bone morphogenetic protein 2 (BMP2), which is known for its osteoinductive potential and is clinically applied to heal large bone defects in combination with 3D scaffolds. In the human body, HS are bound to core proteins forming HS proteoglycans who are either part of the ECM or appear at the cell surface. The role of extra cellular and cell surface HS together with integrins on BMP2 signaling and cellular adhesion is currently under scientific debate. Few studies suggest that exogenous HS up-regulates BMP2 signaling whereas cell surface HS negatively influences BMP2 bioactivity. Further, BMP2 induces integrin-mediated cellular adhesion and cell surface HS mediates cell adhesion to fibronectin. However, it is not known how integrins influence BMP2 signaling or how they interact together with immobilized HS and adsorbed BMP2 with respect to osteogenic differentiation. Further, the relation between exogenous and cell surface HS is rarely addressed. We respond to these questions by engineering 2D biomimetic model surfaces based on a streptavidin monolayer, which allows co-immobilizing biotinylated molecules of interest and presenting them to cells. The adhesion ligand cRGD is co-functionalized with oriented HS to which BMP2 adsorbs and the areal mass densities and binding kinetics are precisely characterized ex situ using surface sensitive techniques such as spectroscopic ellipsometry (SE) and quartz crystal microbalance. For the first time, we apply fluorescence based image correlation spectroscopy to characterize biomimetic surfaces in situ for molecular density and measure values comparable to ex situ SE. We further develop this technique by combining it with photo bleaching to reveal information about the average number and distribution of fluorophores per molecule to increase the accuracy. To improve the reproducibility and the high content potential of our biomimetic approach, we created an automated protocol to fabricate homogenous biomimetic platforms with a liquid handling robot. Upon user demand, multiple independent conditions of the biomimetic platform can be build-up in parallel inside 96-well plates and allow to study the effect of members of the BMP family in different concentrations adsorbed to iHS. First, we find that β1- and β3-integrins are both fundamental for the BMP2-SMAD signaling and ALP expression in C2C12 cells, markers for early BMP2 activity and osteogenic differentiation, respectively. Also, BMP2 bound to exogenous HS enhances osteogenic differentiation compared to BMP2 directly immobilized on the platform and HS sustains BMP2 activity. We have further evidence that BMP2 adsorbed on HS is more bioactive than BMP4, BMP6 and BMP7 regarding early BMP2 signaling. By plating CHO wild type cell and those with a cell surface HS deficiency, we reveal a positive effect of csHS on BMP2 signaling at low aBMP2 doses, which is in contradiction to results with higher doses in literature. Finally we show that csHS has a negative effect on cell spreading on cRGD surfaces and even more on surfaces presenting cRGD and exogenous HS. We conclude that automatically fabricated, precisely characterized and highly flexible biomimetic streptavidin platforms are a great tool to study the combinatorial influence of growth factors, GAGs and peptides on cell fate.

Keywords:
Heparan sulfate, BMP2, Integrins, Automation, Surface characterization, Biomimetics