Three-dimensional bioprinting uses additive manufacturing techniques for the automated fabrication of hierarchically organized living constructs. The building blocks are often hydrogel-based bioinks, which need to be printed into structures with high shape fidelity to the intended computer-aided design. For optimal cell performance, relatively soft and printable inks are preferred, although these undergo significant deformation during the printing process, which may impair shape fidelity. While the concept of good or poor printability seems rather intuitive, its quantitative definition lacks consensus and depends on multiple rheological and chemical parameters of the ink. This review discusses qualitative and quantitative methodologies to evaluate printability of bioinks for extrusion- and lithography-based bioprinting. The physicochemical parameters influencing shape fidelity are discussed, together with their importance in establishing new models, predictive tools and printing methods that are deemed instrumental for the design of next-generation bioinks, and for reproducible comparison of their structural performance.

Biofabrication technologies, including stereolithography and extrusion‐based printing, are revolutionizing the creation of complex engineered tissues. The current paradigm in bioprinting relies on the additive layer‐by‐layer deposition and assembly of repetitive building blocks, typically cell‐laden hydrogel fibers or voxels, single cells, or cellular aggregates. The scalability of these additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality. Overcoming such limitations, the volumetric bioprinting of clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds is described. An optical‐tomography‐inspired printing approach, based on visible light projection, is developed to generate cell‐laden tissue constructs with high viability (>85%) from gelatin‐based photoresponsive hydrogels. Free‐form architectures, difficult to reproduce with conventional printing, are obtained, including anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts. The latter undergoes maturation in vitro as the bioprinted chondroprogenitor cells synthesize neo‐fibrocartilage matrix. Moreover, free‐floating structures are generated, as demonstrated by printing functional hydrogel‐based ball‐and‐cage fluidic valves. Volumetric bioprinting permits the creation of geometrically complex, centimeter‐scale constructs at an unprecedented printing velocity, opening new avenues for upscaling the production of hydrogel‐based constructs and for their application in tissue engineering, regenerative medicine, and soft robotics.

Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.

Organ‐ and tissue‐level biological functions are intimately linked to microscale cell–cell interactions and to the overarching tissue architecture. Together, biofabrication and organoid technologies offer the unique potential to engineer multi‐scale living constructs, with cellular microenvironments formed by stem cell self‐assembled structures embedded in customizable bioprinted geometries. This study introduces the volumetric bioprinting of complex organoid‐laden constructs, which capture key functions of the human liver. Volumetric bioprinting via optical tomography shapes organoid‐laden gelatin hydrogels into complex centimeter‐scale 3D structures in under 20 s. Optically tuned bioresins enable refractive index matching of specific intracellular structures, countering the disruptive impact of cell‐mediated light scattering on printing resolution. This layerless, nozzle‐free technique poses no harmful mechanical stresses on organoids, resulting in superior viability and morphology preservation post‐printing. Bioprinted organoids undergo hepatocytic differentiation showing albumin synthesis, liver‐specific enzyme activity, and remarkably acquired native‐like polarization. Organoids embedded within low stiffness gelatins (<2 kPa) are bioprinted into mathematically defined lattices with varying degrees of pore network tortuosity, and cultured under perfusion. These structures act as metabolic biofactories in which liver‐specific ammonia detoxification can be enhanced by the architectural profile of the constructs. This technology opens up new possibilities for regenerative medicine and personalized drug testing.

Infections caused by biofilm-forming bacteria are a major threat to hospitalized patients and the main cause of chronic obstructive pulmonary disease and cystic fibrosis. There is an urgent necessity for novel therapeutic approaches, since current antibiotic delivery fails to eliminate biofilm-protected bacteria. In this study, ciprofloxacin-loaded poly(lactic-co-glycolic acid) nanoparticles, which were functionalized with DNase I, were fabricated using a green-solvent based method and their antibiofilm activity was assessed against Pseudomonas aeruginosa biofilms. Such nanoparticles constitute a paradigm shift in biofilm treatment, since, besides releasing ciprofloxacin in a controlled fashion, they are able to target and disassemble the biofilm by degrading the extracellular DNA that stabilize the biofilm matrix. These carriers were compared with free-soluble ciprofloxacin, and ciprofloxacin encapsulated in untreated and poly(lysine)-coated nanoparticles. DNase I-activated nanoparticles were not only able to prevent biofilm formation from planktonic bacteria, but they also successfully reduced established biofilm mass, size and living cell density, as observed in a dynamic environment in a flow cell biofilm assay. Moreover, repeated administration over three days of DNase I-coated nanoparticles encapsulating ciprofloxacin was able to reduce by 95% and then eradicate more than 99.8% of established biofilm, outperforming all the other nanoparticle formulations and the free-drug tested in this study. These promising results, together with minimal cytotoxicity as tested on J774 macrophages, allow obtaining novel antimicrobial nanoparticles, as well as provide clues to design the next generation of drug delivery devices to treat persistent bacterial infections.