Due to current limitations in experimental models, we still lack a general understanding of how cells integrate simultaneous chemical and physical stimuli into a specific output. In addition to the well-studied effect of growth factors, signaling- and adhesion-molecules, over the last decade it has been recognized that physical properties of the extracellular environment also have a profound influence on cell behavior and differentiation. These physical stimuli include mechanical stiffness and topography of the cellular environment, as well as spatial patterns of ligand presentation. The overall aim of project E2 is to manipulate cell behavior and differentiation via biochemically and mechanically tailored nanostructured two-dimensional (2D) substrates and three-dimensional (3D) scaffolds.
The geometry of ligand presentation spans different length scales, ranging from nanoscale distributions at the cell membrane to several micrometers for the arrangement of single sites of adhesion at the cellular level. In addition, graded distributions of cell-adhesion molecules and signaling factors play major roles during development and regeneration of multicellular organisms. Microcontact printing (µCP) or microfluidic networks (µFN) are employed to reliably produce regular patterns or gradients of substrate-bound biomolecules (see subproject E2.4: Cell Adhesion and Migration on Micro- and Nanostructured Substrates). For membrane bound proteins, a prerequisite for achieving the desired biological effects of a surface-tethered ligand is its correct orientation as well as a suitable surface-ligand separation. Suitable methods to bio-functionalize surfaces with proteins in a specific orientation and conformation therefore need to be worked out (see subproject E2.2: Nanostructured Templates with Cadherin Specific Adhesive Properties). A future challenge in this field is the reliable production of cellular growth-substrates with multiple bio-functionalities that can be spatially controlled.
Forces and Elasticity
In addition to the geometry of ligand presentation, the stiffness of the environment has emerged as a major and formerly overlooked regulator of cell behavior and fate. Cells do not only apply forces to their growth substrate, they also measure elastic modes in their environment and react to them. As a consequence, cell behavior, including stem-cell differentiation, changes dramatically as cells are grown on increasingly softer substrates. Hence, a profound understanding on the influence of elasticity on cell behavior not only depends on the fabrication and calibration of flexible growth substrates (see subproject E2.3: Behavior of Cells in 2D and 3D Micro- and Nanostructured Patterns) but also on precise measurements of cell adhesion forces on bio-functionalized surfaces (see subproject E2.4: Cell Adhesion and Migration on Micro- and Nanostructured Substrates).
Three-Dimensional Growth Substrates
Cells grown on 2D tissue culture substrates often differ considerably from those grown in more physiological 3D environments regarding their morphology, cell-cell and cell-matrix interactions, and cell differentiation. In vitro 3D models currently used include scaffolds fabricated from purified ECM molecules and synthetic biomaterials. However, the geometry and physical properties of these scaffolds are difficult to control. In a collaborative approach, direct laser writing (DLW, also see subproject A1.4: Three-Dimensional Photonic Crystals) has successfully been applied to realize three-dimensional “designer Petri-dishes” for the study of cell growth (see subproject E2.3: Behavior of Cells in 2D and 3D Micro- and Nanostructured Patterns). The long-term goal of this project is to realize flexible 3D templates with multiple bio-functionalities, e.g., adhesive RGD-peptides or ECM-proteins in combination with signaling molecules like cadherins or ephrins. For that purpose, flexible monomeric as well as polymeric materials decorated with wavelength specific photo-click functionalities will be developed in the group of Christopher Barner-Kowollik (see subproject E2.6: Polymer Surface Modification for Targeted Cell Attachment), which can react rapidly in a spatially highly resolved fashion with suitably functionalized biomarkers as counterparts in the photo-induced ligation chemistries. These novel materials will allow for systematic studies of the effects of spatial ligand distributions and mechanical scaffold stiffness on cell behavior and stem cell differentiation in 3D environments.