Rheolution Article | December 2022
What is FRESH bioprinting and how it works?
by Gloria Pinilla, M.Sc.
Senior Application Specialist, Rheolution Inc.
Have you ever day-dreamed of the science-fiction idea of treating a patient’s injuries in a floating rejuvenation tank? The phenomenon that allows for this suspension, neutral buoyancy, seems to have inspired researchers to discover a solution for 3D printing materials with soft and liquid-like inks that would have great difficulty maintaining their print fidelity in air, with gravity forces collapsing it. Despite the fact that water-based support fluids (as the ones used in the floatation tanks) would never work with the water-based hydrogels widely used in tissue engineering applications, this idea of 3D printing in a support bath evolved to minimize the effects of gravity by using gel-like materials that could reversibly show solid and liquid-like behaviors, allowing for hydrogel deposition within it.
This month of November, we explored Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting, an embedded bioprinting approach specifically optimized to support the printing of soft and low viscosity liquid-like bioinks, which are widely used for tissue engineering applications. Generally speaking, one can think about embedded 3D bioprinting as a build chamber filled with a support material within which biomaterials and cells are deposited in the 3D space using a syringe-based extruder. The FRESH method then allows the printing of unmodified biological hydrogels such as collagen type I and decellularized ECM with high density cell-laden bioinks for fabrication of functional engineered tissues.
The key engineering challenge in this approach is to develop a support bath material able to hold the soft printed structures as they are extruded and cured while still allowing for the easy movement of the extruder needle through the support bath during printing. Specifically, the rheological requirement for the support bath material is that it must act as a solid below a threshold of applied shear stress, i.e. the yield stress, at which it transitions from solid to liquid-like behavior. Around the needle, the liquid-like behavior of the support bath (applied shear stress higher than the yield stress) allows the easy movement of the needle. Once the needle departs, the support bath resolidifies (applied shear stress lower than the yield stress). The optimal yield stress for the support bath is dependent on the materials being printed, the needle diameter and length, and process parameters such as the print speed [1].
The FRESH printing method presents several outstanding advantages. First, its versatility. Although FRESH was originally described using a gelatin-based support bath [2], the development of different baths has enabled the printing of soft materials with the broadest range of gelation mechanisms (Figure 1). Moreover, since the cross-linking agents are mixed in the bath, the extruded bioink filament is immediately exposed to the liquid on all sides, which rapidly initiates a concurrent gelation process. This is in contrast to printing in air, where there are limited ways to initiate gelation. Second, the support bath provides an environment that prevents cell death and maintains high cell viability during the printing process. In addition to temperature control, the aqueous part of the support bath can be composed of cell culture media, growth factors or any other biomolecule intended to protect cell survival. As a consequence, the third main advantage of the technique is the size of the constructs that could be printed. To date, most 3D bioprinted tissue constructs have been relatively small when compared to the tissues or organs they are intended to replace. FRESH has already been used to create a neonatal-scale physical model of the human heart out of collagen type I based on MRI imaging data [3]. And researchers affirm that this technique has the potential to be a scalable additive manufacturing technology that can produce full-scale tissues based on patient-specific anatomic data.
Figure 1. Illustration showing the versatility of the FRESH technique, allowing printing of a broad range of bioinks including of soft and low viscosity natural materials, as well as synthetic materials. Additionally, the embedded nature of FRESH enables printing using layer-by-layer, non-planar or freeform print pathing to produce geometries that are not possible when printing in air (modified image from Shiwarsky et al. [1]).
Finally, the majority of current 3D bioprinting techniques, including FRESH, utilize a layer-by-layer printing approach. While the FRESH method itself would allow for true freeform printing, software programs allowing for non-planar printing configurations have been greatly sought after. In our recently published Article of the month, we discussed the terrific work developed by the group of Prof. Jonathan T. Butcher about the development of a novel computational method that allows for slicing 3D digital constructs for non-planar printing. The development of such software provides more flexible and freeform printing, having the potential to advance significantly the field towards the creation of organ-like curved and thin structures, including heart valves or traqueas.
While significant work has been done, the community believes that we are only at the beginning of what the FRESH technique combined with sophisticated software tools can offer researchers in the fields of tissue engineering and 3D biofabrication.
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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.
3D-bioprinting involves printing a biomaterial or 'bioink' that contains cells, forming structures similar to living tissues. The process requires careful optimization to ensure the material holds its shape and function, and that the cells survive the printing process. Current bioprinting techniques, such as extrusion, inkjet and laser-assisted bioprinting, come with their own advantages and limitations. The ultimate goal is to bring in vitro bioprinting into the operating room, overcoming challenges like sterility, regulation, and ethical considerations.
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.
