• D1 - Natural and synthetic hybrid hydrogels to study the effect of mechanical anisotropy on cell behavior and guidance
Mechanobiology in Epithelial
3D Tissue Constructs

Natural and synthetic hybrid hydrogels to study the effect of mechanical anisotropy on cell behavior and guidance

Lead supervisor: L. De Laporte, Co-supervisor: A. Lampert, Junior supervisor: Y. Chandorkar
DWI - Leibniz Institute for Interactive Materials

Hypothesis: Controlled mechanical 3D anisotropy can be used to steer cell behavior.

Key preliminary results of project D1. (A) Aligned nerve cell extensions from a dorsal root ganglion in a PEG-based Anisogel (unpublished). (B) Aligned primary nerve cells in a fibrin-based Anisogelfrom 39. (C) Fibroblast adhering to RGD-modified microgel from74.
Background: To bridge the gap between implantable constructs with complex architectures and injectable isotropic hydrogels, a hybrid hydrogel was developed to enable a low-invasive application while creating an oriented structure after injection. The hybrid hydrogel is known as an Anisogel and consists of 2 components: (i) rod-shaped microgels or short fibers (green objects) that are rendered magnetically responsive by incorporating a low amount of superparamagnetic iron oxide nanoparticles (SPIONs, black dots) into the structures and (ii) a surrounding hydrogel precursor solution (blue network). The anisometric guiding elements align in the presence of a low external magnetic field in the millitesla range, after which the surrounding solution crosslinks to fix the orientation so that the magnetic field can be removed. Initial reports by our group have demonstrated that fibrin-based Anisogels have the ability to align cell and nerve growth with a minimal proportion of guiding elements (2 vol%; B)39, while the nerves inside the Anisogel show spontaneous electrical activity proving neuronal functionality. Importantly, electrical signals propagate along the anisotropy axis of the material93. GRGDS-modified microgels inside a fibrin-based Anisogel further enhance fibroblast alignment and lead to a reduction in fibronectin production, indicating successful replacement of structural proteins. In addition, Yes-associated protein translocation to the nucleus increases with the concentration of microgels, indicating cellular sensing of the overall anisotropic mechanical properties of the Anisogel74.
Recently, the fibrin has been replaced by an engineered synthetic PEG-based hydrogel. The advantage of synthetic gels is that their mechanical, physical, and biochemical properties can be controlled bottom-up. Depending on the functional PEG endgroups, different crosslinking mechanisms can be applied: click reactions or enzymatically induced. The crosslinks contain matrix-metalloproteinase-sensitive peptide sequences to enable cleavage of the hydrogel network on cell demand. The biocompatible chemistries allow for easy incorporation of specific cell adhesive molecules, such as full ECM proteins, tailored ECM fragments
24,94 or peptides, depending on the cell/tissue types to be studied95.

Aims: The main objective is to engineer a 3D PEG-based synthetic hydrogel for the various tissue models in MEƎT. In close collaboration with A1-A3 and B3, the mechanical and biochemical properties and the degradation rate of the hydrogel is optimized for each application. Using established methods, a library of magneto-responsive rod-shaped microgels and short fibers is produced with different dimensions, aspect ratios, chemistries, cell adhesive ligands, stiffness and surface topographies. Cells are mixed inside the hydrogel solution and, after orientation, the effect of the 3D mechanical anisotropy, created by the different material design parameters, are analyzed on epithelial cell migration (A2, A3) and stem cell differentiation (A1, B3).

Approach: Two different techniques are applied to produce the anisometric guiding elements: an in-mold polymerization method to prepare PEG-based microgels and an electrospinning/microcutting technique to prepare polylactide-co-glycolide (PLGA) or poly caprolactone (PCL) short fibers. Their properties and functionality are tested using atomic force and STED microscopy, cell culture experiments and immunostainings. The mechanical properties of the surrounding hydrogel inside the anisogel are optimized via rheology and dynamic mechanical analysis, and specific biological ECM fragments are produced in E. coli in collaboration with U. Schwaneberg (DWI/ABBt RWTH). In close collaboration with A1-A3 and B3, specific cells are incorporated inside or on top of the gels to analyze their behavior with dedicated imaging and electrophysiological techniques.