Advances and Future Perspectives in 4D Bioprinting

Abstract

Three-dimensionally printed constructs are static and do not recapitulate the dynamic nature of tissues. Four-dimensional (4D) bioprinting has emerged to include conformational changes in printed structures in a predetermined fashion using stimuli-responsive biomaterials and/or cells. The ability to make such dynamic constructs would enable an individual to fabricate tissue structures that can undergo morphological changes. Furthermore, other fields (bioactuation, biorobotics, and biosensing) will benefit from developments in 4D bioprinting. Here, the authors discuss stimuli-responsive biomaterials as potential bioinks for 4D bioprinting. Natural cell forces can also be incorporated into 4D bioprinted structures. The authors introduce mathematical modeling to predict the transition and final state of 4D printed constructs. Different potential applications of 4D bioprinting are also described. Finally, the authors highlight future perspectives for this emerging technology in biomedicine.

Keywords: 4D bioprinting; additive manufacturing; bioinks; stimuli-responsive biomaterials; tissue engineering.

Figure 1.

Schematic of different printing technologies (3D, 3D bioprinting, 4D, and 4D bioprinting) using conventional materials, cells, and smart materials. Cells are involved in bioprinting technologies. We defined 4D bioprinting as 3D printing of cell-laden materials in which the printed structures would be able to respond to external stimulus due to stimuli-responsive bioinks or internal cell forces.

Figure 2.

Multimaterial-based ink preparation and deposition process to make electrically conductive and cell-laden structures. i) Schematic diagram for making DNA/HA-coated single-walled CNT inks. ii) Schematic of 3D printing steps to fabricate conductive fibers. iii) Schematic diagram indicating the connection of printed fibers into GelMA hydrogels. iv) Top view of printed fiber incorporated in GelMA hydrogels. v) Encapsulated cardiomyocytes in GelMA hydrogels with 3D stacked CNT fibers on day 10 of culture. Immunostaining was done for sarcomeric α-actinin (green), cell nuclei (blue), and Cx-43 (red). Reproduced with permission from Shin et al. [50].

Figure 3.

Making cell aggregates triggered by magnetic field. (a) The use of intracellular magnetic particles and external magnetic field to control embryonic stem cell aggregation. (b) Photographs (i) and illustration (ii) of stem cell aggregate movement under the influence of magnetic field. Reproduced with permission from Du et al. [58].

Figure 4.

4D biofabrication of cell-laden biomaterials. (i) 4D bioprinting of cell-laden self-folding hydrogel-based tubes using methacrylated alginate (AA-MA) or HA-MA on different substrates (glass or polystyrene (PS)). Green light (530 nm) was used for mild drying of structures. Instant folding into tubes was obtained upon immersion of crosslinked films in water, phosphate-buffered saline (PBS), or cell culture media. (ii) The tube responsiveness (cartoons (upper panel) and representative photographs (lower panel)) in water (1), same tube immersed in CaCl₂ solution (2), which led to an additional crosslinking of alginate with Ca²⁺ ions and complete unfolding of the tube, and folded tube immersed in ethylenediaminetetraacetic acid (EDTA) solution (3), where EDTA bound the Ca²⁺ ions from the alginate, leading to refolding of the film into a tube [119].

Figure 5.

4D printed grippers as biorobots. (a) Multimaterial grippers were fabricated with different designs. (b) The demonstration of the transition between as-printed and temporary shapes of multimaterial grippers. (c) The snapshots of the process of grabbing an object. Reproduced with permission from Ge et al. [101].