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.

Three-dimensional (3D) bioprinting is a promising technology for manufacturing cell-laden tissue-engineered constructs. Larger tissue substitutes, however, require a vascularized network to ensure nutrition supply. Therefore, tailored bioinks combining 3D printability and cell-induced vascularization are needed. We hypothesize that tailored hydrogel blends made of agarose-type I collagen and agarose-fibrinogen are 3D printable and will allow the formation of capillary-like structures by human umbilical vein endothelial cells and human dermal fibroblasts. Samples were casted, incubated for 14 days, and analyzed by immunohistology and two-photon laser scanning microscopy. The 3D printability of the hydrogel blends was examined using a drop-on-demand printing system. The rheological behavior was also investigated. Substantial capillary network formation was observed in agarose-type I collagen hydrogel blends with concentrations of 0.2% or 0.5% collagen and 0.5% agarose. Furthermore, storage moduli of agarose-collagen blends were significantly increased compared to those of the corresponding single components (448 Pa for 0.5% agarose, 148 Pa for 0.5% collagen, and 1551 Pa for 0.5% agarose-0.5% collagen). Neither the addition of collagen nor fibrinogen significantly impaired the printing resolution. In conclusion, we present a tailored hydrogel blend that can be printed in 3D and in parallel exhibits cell-induced vascularization capability.

In recent years, novel biofabrication technologies have enabled the rapid manufacture of hydrogel-cell suspensions into tissue-imitating constructs. The development of novel materials for biofabrication still remains a challenge due to a gap between contradicting requirements such as three-dimensional printability and optimal cytocompatibility. We hypothesise that blending of different hydrogels could lead to a novel material with favourable biological and printing properties. In our work, we combined agarose and type I collagen in order to develop a hydrogel blend capable of long-term cell encapsulation of human umbilical artery smooth muscle cells (HUASMCs) and 3D drop-on-demand printing. Different blends were prepared with 0.25%, 0.5%, 0.75%, and 1.5% agarose and 0.2% type I collagen. The cell morphology of HUASMCs and the printing accuracy were assessed for each agarose-collagen combination, keeping the content of collagen constant. The hydrogel blend which displayed sufficient cell spreading and printing accuracy (0.5% agarose, 0.2% type I collagen, AGR0.5COLL0.2) was then characterised based on swelling and degradation over 21 days and mechanical stiffness. The cellular response regarding cell attachment of HUASMCs embedded in the hydrogel blend was further studied using SEM, TEM, and TPLSM. Printing trials were fabricated in a drop-on-demand printing process. The swelling and degradation evaluation showed an average of 20% mass loss and less than 10% swelling. AGR0.5COLL0.2 exhibited significant increase in stiffness compared to pure agarose and type I collagen. In addition, columns of AGR0.5COLL0.2 three centimeters in height were successfully printed submerged in cooled perfluorocarbon, proving the intrinsic printability of the hydrogel blend. Ultimately, a promising novel hydrogel blend showing cell spreading and attachment as well as suitability for bioprinting was identified and could, for example, serve in the manufacture of in vitro 3D models to capture more complex features of disease and drug discovery.