Abstract3D bioprinting is a novel platform for engineering complex, three-dimensional (3D) tissues that mimic real ones. The development of hybrid bioinks is a viable strategy that integrates the desirable properties of the constituents. In this work, we present a hybrid bioink composed of alginate and cellulose 2 nanocrystals (CNCs) and explore its suitability for extrusion-based bioprinting. This bioink possesses excellent shear-thinning property, can be easily extruded through the nozzle, and provides good initial shape fidelity. It has been demonstrated that the viscosities during extrusion were at least two orders of magnitude lower than those at small shear rates, enabling the bioinks to be extruded through the nozzle (100 μm inner diameter) readily without clogging. This bioink was then used to print a liver-mimetic honeycomb 3D structure containing fibroblast and hepatoma cells. The structures were crosslinked with CaCl 2 and incubated and cultured for 3 days. It was found that the bioprinting process resulted in minimal cell damage making the alginate/CNC hybrid bioink an attractive bioprinting material.Graphical abstract

Abstract3D bioprinting of living cellular constructs with heterogeneity in cell types and extra cellular matrices (ECMs) matching those of biological tissues remains challenging. Here, we demonstrate that, through bioink material design, microextrusion-based (ME) bioprinting techniques have the potential to address this challenge. A new bioink employing alginate (1%), cellulose nanocrystal (CNC) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) was formulated for the direct printing of cell-laden and acellular architectures. The 135ACG ink displayed excellent shear-thinning behavior and solid-like properties, leading to high printability without cell damage. After crosslinking, the ACG gel can also provide a stiff ECM ideal for stromal cell growth. By controlling the degree of substitution and polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matching that of human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially useful for both fundamental biomedical research and translational tissue engineering.

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Here, a class of ink materials and an embedded 3D printing strategy for the fabrication of macroscale elastic tissue‐mimetic constructs are presented. Novel inks composed of 10 wt% glycidyl methacrylated poly(vinyl alcohol) (PVAGMA) with different degrees of substitution (DOS) and 4 wt% cellulose nanocrystals (CNCs) (PVAGMA(DOS)/CNC) with strong shear‐thinning property are developed. By controlling the DOS of PVAGMA, hydrogels with desired mechanical stiffness mimicking that of healthy and diseased artery are designed to construct vascular phantoms. Cyclic tensile tests and in vitro hemodynamic study performed on phantoms demonstrate their excellent mechanical stability and low hysteresis. The burst pressure is found to be about 273 mmHg for PVAGMA2/CNC and 102 mmHg for PVAGMA4/CNC and the printed vascular phantoms are able to withstand 863K cycles over 10 days. Further, their suitability for ultrasonic imaging is demonstrated. Via B‐mode imaging, it is found that the vessel strains under a pulsatile flow are 11.7 ± 1.0% and 6.5 ± 1.5% for PVAGMA2/CNC and PVAGMA4/CNC vessels, respectively, perfectly matching the behaviors of healthy and atherosclerotic arteries. It exemplifies that the ink materials and printing strategy can have broad applications in biomedical research, especially for the fabrication of complex elastic tissue phantoms.