Despite the widespread use of 3D-bioprinting in tissue engineering and biomedical research, 3D-printed scaffolds still lack precision and biomimetism, as they are developed as static and inanimate materials, whereas the human body is a dynamic environment. With 4D-bioprinting, the scientific community recently integrated “time” as the fourth dimension in the printing process of biomaterials. Developing 4D-scaffolds that have the ability to evolve over time after being printed, is a new challenge. Achieving complex and well-defined shapes with biocompatible biomaterials would allow an adapted tissue engineering for organs with an intricate geometry such as gut villi or pulmonary acini. In regards to specific diseases, patient-individualized heart dressings could for instance also be developed.
A group of researchers from Alsberg’s Lab led by prof Eben Alsberg, part of the Richard and Loan Hill Department of Biomedical Engineering in the University of Illinois Chicago, USA, developed in 2012 a new promising material for tissue engineering using oxidized and methacrylated alginate (OMA). The methacrylation degree of alginate allows to tune the mechanical properties of the final hydrogel, but also to control the amount of hydrolyzable spots (i.e. spots sensitive to biodegradation) in crosslinked alginate. Since the in vivo biodegradation of such methacrylated crosslinked alginate is slower than in vitro, this team oxidized the alginate prior to methacrylation in order to change the conformation of the alginate backbone and make it more vulnerable to hydrolysis. Alsberg’s group also previously demonstrated that such biodegradable OMA microgels are cryopreservable for long-term storage with highly viable and functional recovered encapsulated cells: a feature particularly adapted for clinical on-demand applications. In their study entitled Jammed Micro-Flake Hydrogel for Four-Dimensional Living Cell Bioprinting published in the journal Avanced Materials, the authors developed a cytocompatible cell-laden bioink using OMA as a microgel for 4D living cell bioprinting with complex geometries. By creating a cross-linking gradient with a photoinitiator and an UV absorber inside the 3D-printed scaffold, Ding et al. were able to define cell-seeded complex geometries, aiming for a cartilage-like tissue as a proof-of-concept.
Alginate was ionically crosslinked by adding OMA drop by drop into a 0.2 M Ca2+ solution. The obtained precursor beads were blended, stored in 70% ethanol and subsequently resuspended in a photoinitiator and UV absorber solution. During this process, precursor beads turned into flakes with a heterogeneous morphology of 41.7 ± 19.8 µm. This jammed micro-flaked hydrogel, or microgel, displayed shear-thinning and rapid self-healing properties that allowed smooth printing into stable 3D bioconstructs. Before printing, the microgel was mixed with a cell suspension, thus producing the bioink, which was used to print first a simple filament, then more complex patterns such as 4-arm star, 6-arm star, grids, etc. That is where the fourth dimension comes in. Once printed, these constructs can be further photo-crosslinked into 3D-scaffolds with sophisticated geometries using the properties of the photoinitiator and the UV absorber. The former allows to create chemical crosslinking points, when the latter actually absorb the energy coming from the light irradiation, so the light does not get through the whole hydrogel. This process generates a crosslinking gradient that, once the hydrogel is embedded in a specific medium, will induce the bending of the whole structure due to differential hydrogel swelling resulting from the differences in microgel crosslinking densities.
This way, the 4-arm star and the 6-arm star initially printed, can grow into “pseudo-four petal” and “pseudo-six petal” flowers, respectively. Either by using mask-based photolithography or just by orienting the light source in a specific direction on the 3D-printed scaffolds, Alsberg’s team was also able to induce anisotropy in different directions, also forming a “biohelix”, a “bioS” and “kirigami-based” structures e.g. a self-curled structure similar to a human rib cage or a “net-tube”.
In addition to programmable deformation and to this differential hydrogel swelling, the authors observed the ability of alginate to shrink or swell differently with pH stimulation in such 4D-bioconstructs. Depending on the pore size, the carboxylic groups of alginate are indeed more or less accessible. In this study, if we consider for instance a printed OMA microgel filament bended in growth medium, the outer part of the bended filament – the low crosslinking side – will have larger pores. If this filament is immersed in acidic pH, the protons will therefore diffuse faster in the outer part of the gel, which will shrink faster. This reversible process could have an application in tissue engineering e.g. for specific gastric environments.
The cytocompatibility of this 4D-system was confirmed with fibroblasts (NIH3T3), a cancer cell line (HeLa) and human bone-marrow-derived mesenchymal stem cells (hMSC). Chondrogenesis of hMSC was monitored over 21 days with 4D hMSC-laden hydrogel bars cultured in chondrogenesis media. As expected, the photo-induced gradient made the bar change into a C-shape, with HMSC expressing a round morphology, a high viability checked with a live&dead assay, and an increasing production of DNA and phenotypic glycosaminoglycanes (GAG), typical of chondrogenic cells. The authors showed that this shape-shifting property does not impact the proliferation and differentiation of pre printing-embedded cells.
Overall, Ding et al. were able to fabricate 4D bioconstructs with dynamic shape shifting capabilities and complex configurations that enable the potential to biomimic the conformational changes occurring during tissue development and healing. The 4D bioink used in this study – made of encapsulated stem cells in a modified alginate microgel – supports new tissue formation with high printing resolution and high printing fidelity. This morphodynamic biomaterial is promising for tissue engineering and developmental processes.
 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.
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