Microgel-based Regenerative Materials and Biofunctionalization

Gehlen, David Benedikt; De Laporte, Laura (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Over the last decades, significant progress has been made in the field of implants and medical devices, which are capable of taking over the function of essential organs and tissues. In addition, new biomaterial systems are being designed to better understand cell behavior for basic research. However, controlling the cell-material interaction remains a crucial challenge for these systems and devices. For example, in the case of polydimethylsiloxane (PDMS), which is often used to study mechanobiology, most of the coating methods to enable stable cell adhesion, are complex, time-consuming, unstable over time, and include toxic compounds. Therefore, I developed a completely novel method using an anchor peptide with a fused cell adhesive peptide for coating PDMS surfaces in a one-step process in a robust and environmental-safe manner resulting in stable cell adherence. Importantly, one of the main goals in modern medicine remains the production and regeneration of fully functional tissues and organs. A lot of progress has been made over the last decades in designing advanced and smart materials using complex fabrication methods to generate controlled cell constructs. This scientific discipline is called tissue engineering but is still limited due to a large gap with products in clinical use. This is a consequence of the complex nature of native tissues with scales ranging from μm for individual cells, over mm for blood vessels, to cm for arrangements of tubular and other structures. Hydrogels, consisting of either natural or synthetic hydrophilic polymers, are the most promising approach to create injectable 3D scaffolds for tissue regeneration of soft and sensitive tissues. However, high porosity is missing in most synthetic hydrogels with elastic nanometer-size meshes, which is essential for cell infiltration, migration, and proliferation. To overcome this challenge, I developed an easy and rapid process to create granular hydrogel scaffolds. Crosslinked cellulose nanofibril hydrogels with calcium are crushed through a Nylon mesh, leading to homogeneous 3D cell growth in a very effective manner. For a more controlled approach, I developed a microporous scaffold using enzymatically crosslinked microgels. The combination of rod-shaped and spherical microgels results in maximized porosity. For the regeneration of functional tissues or for generating ex vivo models, it is essential to resemble their structure and hierarchical order. For example, the kidney possesses tubular structures. To resemble these tubular structures in an embedded matrix, I produced cellulose nanofibril microgels by pipetting the solution through thin needles into a stirring calcium bath and subsequently breaking them down mechanically during purification. Fibroblasts can be seeded and cultured on these microgels and embedded into synthetic PEG-based hydrogels, which allowed for the production of tubular cell structures inside of a highly controlled surrounding matrix. In vivo therapies for sensitive tissues, such as the spinal cord, require a low invasive injection to avoid implantation and additional removal of healthy tissue. Therefore, I worked on the Anisogel (Anisometric hydrogel) system, which forms an aligned structure in situ after injection with high controllability of the mechanical, biochemical, and structural properties. This is achieved by incorporating superparamagnetic nanoparticles into rod-shaped microgels with defined micron-scale diameters, allowing microgel alignment parallel to a magnetic field in a solution. Next, the solution crosslinks enzymatically to fixate the oriented microgels. Strikingly, I showed for the first time that not only the cells are aligned inside of the Anisogel but also the fibronectin produced by the cells, indicating a potential positive feedback cycle. Additional biofunctionalization of the guiding elements with cell-adhesive peptides results in high interactions with the cells and slightly increased cell-alignment but reduces the amount of fibronectin produced by the cells, which may hinder the regeneration process and replacement of the temporal artificial scaffold during its degradation. Investigating the location of the mechanosensitive protein YAP revealed that the cells are not only aligned by contact guidance but rather feel the overall mechanical anisotropy of the material, indicating a more complex mechanism behind cell alignment. To further study and characterize this, I developed a method with magnet-assemblies for rheology measurements, enabling investigation of the overall mechanical anisotropy of the Anisogel. Furthermore, controlling the Anisogel structure with different dimensions of microgels showed that the volume of guiding elements to initiate nerve alignment can be reduced by using microgels with diameters of 2.5 μm instead of 5 μm. After being able to control all three parameters inside the Anisogel system, which are the mechanical, biochemical, and structural properties, I worked on actively moving the microgels within the surrounding matrix for potential stimulation of cells. However, the embedded microgels did not move and stronger magnetic properties of the microgels are necessary for the future to generate forces in a relevant range. To expand the use of the magneto-responsive microgels for other applications, such as magnetic field-assisted bioprinting, I also developed a method to pre-program the orientation angle of the microgels in a static magnetic field. Therefore, ellipsoidal maghemite nanoparticles were aligned within the microgel network at a defined angle during their crosslinking, enabling the orientation of the final microgels in different directions in the magnetic field according to the defined angle of the pre-aligned ellipsoidal nanoparticles. In conclusion, controlling and mimicking the biochemical properties, macro-porosity, and oriented structures of native tissues results in a better understanding of 3D cell growth in artificial scaffolds, which can be further developed to stimulate and actuate cells during tissue maturation.