in cell biology, [ 29,30 ] e.g., in cell signaling and protein expression. [31][32][33] For instance, it is reported that shear stress promotes maturation of megakaryocytes. [ 34 ] Moderate shear stress was found to have an infl uence on stem cell differentiation. [ 35 ] Excessive shear stress, in contrast, even dispatches cells by disrupting the membrane. These phenomena are even more crucial in bioprinting processes, where hydrogels of high viscosity and small nozzles are applied in an attempt to improve the fi nal printing resolution. Here, we show that both hydrogel viscosity and nozzle size directly affect shear stress. To prevent adverse cell response and printing-related cell death, it is essential to control the shear stress level, identify its most important drivers, and study the cell response upon different stress levels. We hypothesize that regulating shear stress and elucidating its impact would be of great use in balancing cell integrity and printing resolution.We present a microvalve-based bioprinting system for the manufacturing of high resolution, multi-material 3D structures ( Scheme 1 A). Applying a straightforward fl uid-dynamics model, we were able to precisely control shear stress at the nozzle site, which could be adjusted by varying the printing pressure, hydrogel viscosity, and the nozzle diameter. Using this system, we conducted a broad study on how cell viability and proliferation potential are affected by different levels of shear stress (Scheme 1 B). Generating complex, multi-material 3D-structures, we demonstrate that high-resolution printing at moderate, cell-friendly nozzle shear stress are not mutually exclusive.The printer used throughout this study comprised four microvalve-based print heads, each individually controllable and heatable, mounted to a three-axis robotic system ( Figure S1, Supporting Information). A metal stage that could be lowered into a container fi lled with a bi-phasic support liquid-perfl uorocarbon (PFC) and an aqueous crosslinker solution-was used as a printing platform that allowed for the manufacturing of macroscopic, multi-layered 3D structures ( Figure S1, Supporting Information). The presented printing system dispenses single drops of cell-hydrogel suspension by jetting using electromagnetic microvalves. Thus, cells are primarily exposed to mechanical stress in the form of shear stress. To describe the shear stress condition in the nozzle of the valve, we developed a fl uid dynamics model for transient fl ow of non-Newtonian fl uids (hydrogels) based on the Bernoulli equation for unsteady fl ow: Equation (

Recent advances in the field of bioprinting have led to the development of perfusable complex structures. However, most of the existing printed vascular channels lack the composition or key structural and physiological features of natural blood vessels or they make use of more easily printable but less biocompatible hydrogels. Here, we use a drop-on-demand bioprinting technique to generate in vitro blood vessel models, consisting of a continuous endothelium imitating the tunica intima, an elastic smooth muscle cell layer mimicking the tunica media, and a surrounding fibrous and collagenous matrix of fibroblasts mimicking the tunica adventitia. These vessel models with a wall thickness of up to 425 µm and a diameter of about 1 mm were dynamically cultivated in fluidic bioreactors for up to three weeks under physiological flow conditions. High cell viability (>83%) after printing and the expression of VE-Cadherin, smooth muscle actin, and collagen IV were observed throughout the cultivation period. It can be concluded that the proposed novel technique is suitable to achieve perfusable vessel models with a biofunctional multilayer wall composition. Such structures hold potential for the creation of more physiologically relevant in vitro disease models suitable especially as platforms for the pre-screening of drugs.

The mechanical and physicochemical effects of three-dimensional (3D) printable hydrogels on cell behavior are paramount features to consider before manufacturing functional tissues. We hypothesize that besides good printability and cytocompatibility of a supporting hydrogel for the manufacture of individual tissues, it is equally essential to consider beforehand the desired tissue (bone, cartilage, fat). In light of its application, the structure and stiffness of printable hydrogel matrices influence cell geometry, which in turn impacts the differentiation fate. Embedded human mesenchymal stromal cells in printable type I collagen- and chitosan-agarose blends were induced to differentiate toward osteoblasts and adipocytes. Hydrogels' printability in air versus submerged printing in perfluorocarbon was evaluated according to the height, diameter, uniformity, and stability of 3D printed vertical cylinders. Bipotent differentiation within hydrogels was assessed histologically (morphology, cellularity), by immunohistochemistry (vimentin, smooth muscle actin), two-photon microscopy (spatial distribution), and real-time polymerase chain reaction (ALP, BGLAP, OPN, RUNX2, COL 1, aP2, PPARγ-2). Agarose and agarose blends revealed the most valid printability properties by generating uniform cylinders with an average height of 4 mm. Osteogenic differentiation was preferably achieved in anisotropic soft collagen-rich substrates, whereas adipogenic differentiation mostly occurred in isotropic stiff agarose-rich matrices. The conjugation of type I collagen to agarose with varying ratios is possibly a suitable bioink for a broad range of 3D printed mesenchymal tissues.

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.