Bicontinuous aqueous two-phase systems based on GelMA and dextran for tailored porous hydrogels in 3D Tissue Engineering

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In 3D tissue engineering, cells are embedded in cytocompatible hydrogels and processed by casting or bioprinting. Within the 3D structure, the post-processing cellular behavior is determined mainly by the hydrogels' microstructure. Conventional hydrogels are often limited in their tunability, in particular regarding their pore size and structure, and thus encapsulated cells are restricted in proliferation, spreading, migration, and differentiation. To address these shortcomings, we synthesize and investigate novel hydrogel blends based on an aqueous two-phase system (ATPS) consisting of gelatin methacryloyl (GelMA) and dextran. In proof-of-principle experiments, we synthesized homogeneous, regular disconnected-porous and bicontinuous interconnected-porous gels by finely tuning the phase separation mechanism and kinetics of the ATPS solution before ultraviolet (UV) light crosslinking of the GelMA phase and washing out the dextran phase. We showed in preliminary experiments that the hydrogels' pore characteristics were stable after printing. The bicontinuous ATPS hydrogel was suitable for growing several exemplary phenotypically different cell types: Human mesenchymal stem cells, periodontal ligament fibroblasts, and human neuroblastoma cancer cells all maintained optimal viability and expected morphology after seven days of cultivation. Thus, there is a great motivation and necessity to understand the underlying physico-chemical principles that determine the phase separation and structure formation processes in aqueous mixtures of polyampholytes and neutral polysaccharides finally leading to hydrogels with controlled microstructure. In addition, we will examine the effect of the 3D printing process, in particular the printing-induced shear stress on phase separation, the final hydrogel microstructure and their mechanical properties. The affinity of different cell types to grow in ATPS hydrogels will be investigated by evaluating their behavior in both cast and printed hydrogels with bicontinuous microstructure. Furthermore, we want to understand the partitioning of chemoattractants within ATPS solutions and gels. Finally, cell-laden ATPS solutions will be investigated concerning their phase separation mechanisms and the cellular response to both, (bio)printing and post-printing UV-crosslinking will be studied. The insights gained upon these investigations will allow understanding the dominating (macro)molecular interactions and enable the design of extracellular matrix-simulating 3D structures with high-level biomimicry holding the potential to open new opportunities and applications for tissue engineering and regenerative medicine.