Synthese sowie rheologische, mechanische und zellbiologische Charakterisierung von Agarose-Kollagen-Hydrogel-Mischungen für den 3D-Druck von prävaskularisiertem Luftröhren-Ersatzgewebe

The objective of the thesis was the synthesis and characterization of hydrogel blends made of agarose and type I collagen as potential bioinks for the application of drop-on-demand microvalve-based 3D bioprinting of a pre-vascularized trachea substitute. The hydrogel blends were characterized mechanically, rheologically, as well as cell biologically. The hydrogel blends’ printability was evaluated by using a drop-on-demand printing system. In cell culture experiments, the viability, the morphology and the angiogenic potential of hMSC, HUASMC, and HUVEC were assessed in different combinations of agarose and collagen. Furthermore, the effect of printing-induced shear stress on the cell function was investigated. The addition of collagen to agarose significantly improved the mechanical properties of the hydrogel blends. In all materials high cell viability and good metabolic activity were observed for hMSC and HUASMC. It was shown in a co-culture experiment including hMSC and HUVEC that the blended hydrogels promote the formation of capillary-like networks in 3D culture. Printing-related shear stress reduced the cell viability after printing about 20 %. However, in subsequent 2D culture cells recovered and showed normal proliferation and even improved metabolic activity. In 3D culture endothelial cells showed reduced formation of capillary-like networks in samples that were initially exposed to high shear stresses. The agarose-collagen blend with best bioactivity showed only insufficient 3D printing resolution. In future, this limitation could be addressed by incorporating a supporting structure made of a suitable cartilage substitute material that mimics the mechanical properties of the airway

GelMA-collagen blends enable drop-on-demand 3D printablility and promote angiogenesis

Effective vascularization is crucial for three-dimensional (3D) printed hydrogel-cell constructs to efficiently supply cells with oxygen and nutrients. Till date, several hydrogel blends have been developed that allow the in vitro formation of a capillary-like network within the gels but comparatively less effort has been made to improve the suitability of the materials for a 3D bioprinting process. Therefore, we hypothesize that tailored hydrogel blends of photo-crosslinkable gelatin and type I collagen exhibit favorable 3D drop-on-demand printing characteristics in terms of rheological and mechanical properties and that further capillary-like network formation can be induced by co-culturing human umbilical vein endothelial cells and human mesenchymal stem cells within the proposed blends. Gelatin was methacrylated (GelMA) at a high degree of functionalization, mixed with cells, type I collagen, and the photoinitiator Irgacure 2959 and then subsequently crosslinked with UV light. After 14 d of incubation, cells were immunofluorescently labeled (CD31) and displayed using two-photon laser scanning microscopy. Hydrogels were rheologically characterized and dispensable droplet volumes were measured using a custom built 3D drop-on-demand bioprinter. The cell viability remained high in controllable crosslinking conditions both in 2D and 3D. In general, higher UV light exposure and increased Irgacure concentration were associated with lower cell viabilities. Distinctive capillary-like structures were formed in 3D printable GelMA-collagen hydrogels. The characteristic crosslinking time for GelMA in the range of minutes was not altered when GelMA was blended with type I collagen. Moreover, the addition of collagen led to enhanced cell spreading, a shear thinning behavior of the hydrogel solution and increased the storage modulus of the crosslinked gel. We therefore conclude that GelMA-collagen hydrogels exhibit favorable biological as well as rheological properties which are suitable for the manufacturing of pre-vascularized tissue replacement by 3D bioprinting

Silk Fibroin as Adjuvant in the Fabrication of Mechanically Stable Fibrin Biocomposites

Fibrin is a very attractive material for the development of tissue-engineered scaffolds due to its exceptional bioactivity, versatility in the fabrication, affinity to cell mediators; and the possibility to isolate it from blood plasma, making it autologous. However, fibrin application is greatly limited due to its low mechanical properties, fast degradation, and strong contraction in the presence of cells. In this study, we present a new strategy to overcome these drawbacks by combining it with another natural polymer: silk fibroin. Specifically, we fabricated biocomposites of fibrin (5 mg/mL) and silk fibroin (0.1, 0.5 and 1% w/w) by using a dual injection system, followed by ethanol annealing. The shear elastic modulus increased from 23 ± 5 Pa from fibrin alone, to 67 ± 22 Pa for fibrin/silk fibroin 0.1%, 241 ± 67 Pa for fibrin/silk fibroin 0.5% and 456 ± 32 Pa for fibrin/silk fibroin 1%. After culturing for 27 days with strong contractile cells (primary human arterial smooth muscle cells), fibrin/silk fibroin 0.5% and fibrin/silk fibroin 1% featured minimal cell-mediated contraction (ca. 15 and 5% respectively) in contrast with the large surface loss of the pure fibrin scaffolds (ca. 95%). Additionally, the composites enabled the formation of a proper endothelial cell layer after culturing with human primary endothelial cells under standard culture conditions. Overall, the fibrin/silk fibroin composites, manufactured within this study by a simple and scalable biofabrication approach, offer a promising avenue to boost the applicability of fibrin in tissue engineering

Combination of vascularization and cilia formation for three-dimensional airway tissue engineering

Tissue engineering is a promising approach to treat massive airway dysfunctions such as tracheomalacia or tumors. Currently, there is no adequate solution for patients requiring the resection of more than half of the length of their trachea. In this study, the best conditions for combination of three different cell types from the respiratory airway system were investigated to develop a functional ciliated and pre-vascularized mucosal substitute in vitro. Primary human fibroblasts were combined with respiratory epithelial cells and endothelial cells. As scaffolds, fibrin gel and agarose-type I collagen blends were used and cultured with different medium compositions to optimize both vascularization and differentiation of the respiratory epithelium. A mixture of endothelial growth medium and epithelial differentiation medium was shown to optimize both vascularization and epithelial growth and differentiation. After 28 days of co-culture, significantly increased formation of capillary-like structures was observed in fibrin gels with more than three times higher structure volumes compared to agarose-collagen gels. After 35 days, epithelial differentiation into a pseudostratified epithelium with typical marker expression was improved on fibrin gels. While cilia formation was shown on both scaffolds, a higher number of ciliated cells and longer cilia were observed on fibrin gels. The data elucidate the important interplay of co-culture parameters and their impact on vascularization as well as epithelium development and provide a basis for development of functional three-dimensional airway constructs