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 (

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.

Corneal transplantation is the treatment of choice for patients with advanced corneal diseases. However, the outcome may be affected by graft rejection, high associated costs, surgical expertise, and most importantly the worldwide donor shortage. In recent years, bioprinting has emerged as an alternative method for fabricating tissue equivalents using autologous cells with architecture resembling the native tissue. In this study, we propose a freeform and cell‐friendly drop‐on‐demand bioprinting strategy for creating corneal stromal 3D models as suitable implants. Corneal stromal keratocytes (CSK) were bioprinted in collagen‐based bioinks as 3D biomimetic models and the geometrical outcome as well as the functionality of the bioprinted specimens were evaluated after in vitro culture. We showed that our bioprinting method is feasible to fabricate translucent corneal stromal equivalents with optical properties similar to native corneal stromal tissue, as proved by optical coherence tomography. Moreover, the bioprinted CSK were viable after the bioprinting process and maintained their native keratocyte phenotypes after 7 days in in vitro culture, as shown by immunocytochemistry. The proposed bioprinted human 3D corneal models can potentially be used clinically for patients with corneal stromal diseases.