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

在3D组织工程中，细胞被植入细胞相容的水凝胶中，并通过铸造或生物打印进行处理。在3D结构中，后期处理的细胞行为主要由水凝胶的微观结构决定。传统的水凝胶往往在可调性方面受到限制，特别是在孔大小和结构方面，因此微囊化的细胞在增殖、扩散、迁移和分化方面受到限制。为了克服这些缺点，我们合成并研究了基于明胶甲基丙烯酰基(GelMA)和葡聚糖组成的双水相体系的新型水凝胶共混物。在原理验证实验中，我们通过微调ATPS溶液的相分离机理和动力学，然后对GelMA相进行紫外光交联，并洗去葡聚糖相，合成了均匀的、规则的断开孔和双连续的互连孔凝胶。初步实验表明，打印后的水凝胶具有稳定的孔道特性。双连续的ATPS水凝胶适合于培养几种典型的表型不同的细胞类型：人间充质干细胞、牙周膜成纤维细胞和人神经母细胞瘤癌细胞在培养7天后都保持了最佳的活性和预期的形态。因此，很有必要了解决定两性聚体和中性多糖水溶液的相分离和结构形成过程的基本物理化学原理，最终形成具有可控微结构的水凝胶。此外，我们还将研究3D打印过程，特别是打印引起的剪切应力对相分离、最终水凝胶微观结构及其力学性能的影响。不同细胞类型在ATPS水凝胶中生长的亲和力将通过评估它们在具有双连续微结构的铸造型和印刷型水凝胶中的行为来研究。此外，我们还想了解化学诱导剂在ATPS溶液和凝胶中的分配情况。最后，我们将研究细胞负载的ATPS溶液的相分离机理，以及细胞对(生物)打印和打印后UV交联的响应。从这些研究中获得的见解将使我们能够理解主要的(宏观)分子相互作用，并能够利用高水平的生物仿生来设计模拟细胞外基质的3D结构，从而为组织工程和再生医学开辟新的机会和应用。

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.