3D Lithographical Scaffolds for Stem Cell Differentiation

A pre-requisite for cell differentiation in a 3D environment with subsequent tissue formation is a coordinated contact formation, together with defined interactions and communication between the cells. In order to study cellular interactions, one is currently limited to sectioning dead tissue or to ex- vivo co-culture. The static aspect of non-living cells allows no dynamic view, and in a co-culturing approach within a provided matrix, cells are embedded randomly. Hence, cell-cell contacts form at arbitrary areas within the 3D matrix. Therefore, analysis of molecular inter-population interactions is more difficult. Moreover, culturing of cells restricted to two dimensions is known to alter the cellular pheno/genotype yielding different behavior, polarity, protein distribution, and cell signaling. Hence, a system that enables the molecular characterization of cell adhesion, communication and in particular differentiation, like for e.g. during an osteogenesis, is inevitably needed at the single-cell level in the context of tissue formation. The main focus of LiSSCeD will be the development of three-dimensional cell scaffolds mimicking the extracellular matrix. We plan to combine new 3D lithography methods namely multiphoton- and Stimulated-Emission-Depletion (STED)-lithography to structure polymers in three dimensions from hundreds of microns down to several nanometers. Thereby, a strict size control of the polymer nanostructures, their mechanical properties as well as the selective functionalization with biomolecules mediating mesenchymal stem cell (MSC) differentiation will be achieved. The scaffolds will be used as a tool for identification and subsequent optimization of conditions required for MSC osteogenesis. The project consortium covers all research fields relevant in this project, namely multiphoton lithography, single molecule microscopy, localization microscopy as well as cell biophysics (expertise of the Department of Medical Engineering at the University of Applied Sciences). The Institute of Applied Physics at the JKU provides its expertise in STED-lithography (as the only institution in Austria), together with surface- and polymer-chemistry. A close cooperation with the Ludwig Boltzmann Institute for Experimental and Clinical Traumatology (LBI) and the Department of Obstetrics at the Medical University of Vienna, which both have a long standing experience in stem cell research and tissue engineering, will help us in cell cultivation within this project.

Three-dimensional scaffolds are crucial tools for tissue engineering, providing biomimetic environments that support the growth and function of different cell types. However, a challenge is still replicating the intricate geometric, functional, and mechanical properties of tissue in a comprehensive organ-on-a-chip model (e.g. bone tissue). This project focused on developing 3D scaffolds that mimic the ExtraCellular Matrix (ECM) using Multiphoton Lithography (MPL) and STimulated Emission Depletion (STED)-lithography, with feature sizes ranging from micro- to nanometers. These scaffolds enable co-culture of different cell types, allowing for the first-time study of cell-cell and cell-scaffold interactions at the nanoscopic level. Throughout the project, we achieved advancements in developing advanced techniques for efficient photo-grafting of surfaces as well as the fabrication of protein-based polymer 3D scaffolds designed to promote cell differentiation. We developed buffer systems that significantly enhance the efficiency of photochemically induced surface modifications at the single-molecule level. Buffers containing paramagnetic cations and radical oxygen-promoting species improve Laser-Assisted Protein Adsorption by Photobleaching (LAPAP) of fluorescently labeled oligonucleotides or biotin onto 2D and 3D acrylate scaffolds created via MPL. Single-molecule fluorescence microscopy was used to quantify photo-painting efficiency. We utilized Michler's ethyl ketone for STED-inspired MPL, achieving 40nm feature sizes with low visible-range autofluorescence, making it ideal for protein or cell scaffolds. We developed protein-based photoresists (streptavidin, bovine serum albumin) using crosslinkers such as polyethylene glycol diacrylate and methacrylated hyaluronic acid, in combination with a vitamin-based photoinitiator. Additionally, we utilized 2D/3D direct laser writing techniques to fabricate water-soluble protein-based scaffolds with tunable mechanical properties. The resulting scaffolds exhibited Youngs moduli ranging from 80 MPa for artificial polymers to 40 kPa for protein-based materials, with feature sizes below 100 nm. Functional characterization using fluorescently labeled biotin and antibodies confirmed their bioactivity. We leveraged recent advancements to fabricate hybrid scaffolds by integrating artificial polymer-based and protein-based photoresists using MPL. Specifically, the artificial polymer structures comprised BisSR, carboxyethyl acrylates, and methacrylated collagen type I, allowing for precise control over scaffold architecture and composition to enhance tissue engineering applications. These 3D submicron-structured scaffolds (~300 m3) were used to culture mesenchymal stem cells (MSCs), confirming their viability and mechano-transduction. Imaging of MSC actin and vinculin confirmed scaffold compatibility, while MSCs' osteoblast differentiation highlighted the scaffold's potential for bone tissue engineering.