In March and April 2022, we focused our attention on 3D-Bioprinting: the ability to print a biomaterial that contains cells (also called bioink), starting from a digital 3D-model with theoretically any desired shape. In addition to meeting the specifications of a biomaterial designed for conventional biomedical application – i.a. biocompatibility, biodegradability, and structural integrity – 3D-Bioprinting is greatly dependent on parameters such as the viscosity and crosslinking kinetic of the biomaterial. These two parameters define both (i) printing speed and (ii) printing resolution and thus the ability of the biomaterial to hold the given shape and to fulfill its desired function. This printability requires proper optimization to ensure a reproducible bioprinting process. Regardless of the printing technique used, the key parameter is the cell viability, which is impacted during the printing process. Cells must not only survive the printing process, but also perform their essential function in the tissue construct.
Our previous publication Article of the Month on 3D-Bioprinting presented the development of a composite bioink made of collagen and hyaluronan, seeded with human bone marrow-derived mesenchymal stem cells (hMSC). Anisotropic 3D-scaffolds assessing the multi-scale organization of native tissue were extruded for the regeneration of cartilage-like tissues. In our application note entitled “Testing the viscoelasticity of 3D printed hydrogels using ElastoSens™ Bio”, two commercial polymers – silicon and poloxamer – were extruded directly inside the ElastoSens™ Bio’s sample holders. The impact of printed volume fraction on the viscoelastic properties of the scaffold was established.
The extrusion technique used in both studies is the most commonly used and affordable 3D-Bioprinting technique. It allows the continuous deposition of potentially highly viscous materials containing high cell densities close to physiological densities, while being able to print with a large variety of biomaterials. Cell viability using extrusion bioprinting is known to be lower than with other techniques (40-86%) due to shear stresses inflicted on cells that can disrupt the cell membranes. Using a larger nozzle size will e.g. lower the pressure, but induce a major drawback both in resolution and printing speed. A balance between those parameters has to be found for each application, in order to keep a sufficient printing feasibility with a high enough cell viability, paramount for achieving tissue functionality.
Inkjet bioprinting is a low cost, high resolution and high-speed technology. It relies on acoustic, mechanical/piezo-electric, or thermal stimuli to produce pulses of pressure that force 1-100 pL liquid droplets out of the nozzle. The cell-seeded biomaterial has thus to be liquid, and this is a major drawback for clinical application, as this liquid precursor may leak into neighboring tissues or dilute within body fluids. Nevertheless, it was successfully used e.g. for in situ regeneration of functional skin, as the high resolution allowed the deposition of cells with uniform density throughout the volume of the cutaneous lesion.
Less common and more expensive, the laser-assisted bioprinting (LAB) uses energized infrared pulsed laser to carry droplets of bioinks away from a laser-transparent substrate to the printing surface. LAB allows printing with high cell density, high cell viability (>95%) and high resolution. But it requires rapid gelation kinetics to achieve high shape fidelity, and metallic residues may be present in situ, as the laser-transparent subtract is usually coated with a layer of e.g. gold (Au) or titanium (Ti).
3D-Bioprinting is a growing field of research. Scientists are currently trying to develop new printed alternatives for the whole human body, from the heart, lungs and neural network, to the skin, bone, cartilage, pancreas and liver (non-exhaustive list). As of today, there are still limitations regarding the clinical translation of 3D-Bioprinting. Bioinks must possess unique characteristics, even after going through the printing process itself (that can be quite traumatic), such as in vivo insolubility, structural stability, biodegradability congruent with tissue regeneration, promotion of cell growth/differentiation, and biocompatibility/non-toxicity. The next ambitious step would be to bring in vitro bioprinting to in situ bioprinting – where living tissues would directly be printed into the defect site in the operating room – sets further challenges, such as sterility preservation, alignment with regulatory standards, and ethical considerations.
 Ding, A., Jeon, O., Cleveland, D., Gasvoda, K.L., Wells, D., Lee, S.J., Alsberg, E., (2022). Jammed Micro-Flake Hydrogel for Four-Dimensional Living Cell Bioprinting. Advanced Materials. 34, 2109394.
 Guvendiren, M., Lu, H.D., Burdick, J.A., (2011). Shear-thinning hydrogels for biomedical applications. Soft Matter, 8, 260–272.
 Matai, I., Kaur, G., Seyedsalehi, A., McClinton, A., Laurencin, C.T., (2020). Progress in 3D bioprinting technology for tissue/organ regenerative engineering. Biomaterials, 226, 119536.
 Murphy, S.V., Atala, A., (2014). 3D bioprinting of tissues and organs. Nature Biotechnology. 32, 773–785.
Ever fantasized about sci-fi healing tanks? The principle of neutral buoyancy behind them has sparked a unique solution for 3D printing with soft, liquid-like inks. The Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting technique has been developed to support the printing of these tricky materials, ideal for tissue engineering applications. The secret lies in a cleverly engineered support bath, able to hold the soft structures while permitting extruder needle movement. With versatility, high cell viability, and potential for large construct printing, FRESH opens up a world of possibilities, bringing us a step closer to 3D bioprinting patient-specific tissues based on anatomical data.
Exploring the frontier of 3D printing, Cornell University researchers have enhanced the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method. They introduced a computational approach for non-planar printing, enabling complex curved structures, improving printing accuracy and augmenting the mechanical properties of bioprints.
4D bioprinting introduces "time" as the fourth dimension, enabling printed objects to change over time. Physical stimuli like light, temperature, or magnetism can induce transformations. The bioprinting process can also allow targeted drug release through changes in temperature or pH. Nano-hydrogels can serve as drug carriers, directed by magnetic fields, and can be quickly degraded by enzymes. Furthermore, hydrogels can react to biological signals, aiding in tumor treatment by releasing drugs then biodegrading over time.
4D-bioprinting introduces "time" into the printing process, allowing printed structures to evolve. Researchers from the University of Illinois Chicago used oxidized and methacrylated alginate (OMA) to develop a bioink for 4D bioprinting. They made OMA into precursor beads, then transformed them into a "microgel" that could be easily printed into stable 3D constructs. Mixed with cells, the microgel became a bioink, which was printed into complex structures. The printed constructs could be further crosslinked to create 3D scaffolds that could change shape due to differential swelling, adding the 4th dimension, "time," to the printing process.
Recent advancements in 3D-bioprinting have opened the possibility of creating engineered tissues mimicking human native tissues. However, reproducing the extracellular matrix (ECM) composition and microscopic architecture within the biomaterial ink remains challenging. To tackle this, a research group from the AO Research Institute Davos developed a bioink containing both collagen type 1 and tyramine derivative hyaluronan for tissue engineering. They used optimal concentrations of these two components, along with human bone marrow-derived mesenchymal stromal cells. Two crosslinking pathways were used to induce gelation and create a 3D scaffold that was then printed using the bioink's shear-thinning properties. They successfully printed an anisotropic hydrogel, achieving control over the microscopic organization of the matrix. This breakthrough could pave the way for better mimicry of native human tissues, especially anisotropic ones, in tissue engineering applications.
In January 2022, we studied alginate hydrogels in tissue engineering and drug delivery. Alginate, often used as a bioink in 3D bioprinting, forms hydrogels with tunable properties. We examined the effects of varying CaCl2 concentration on alginate crosslinking, crucial for 3D printing. Due to alginate's low viscosity, printing methods include pre-crosslinking with CaCl2, using a coaxial needle for immediate crosslinking, or mixing alginate with other biomaterials to improve printability.