4D printing of self-folding and cell-encapsulating 3D microstructures as scaffolds for tissue-engineering applications

Abstract

Technology of tissue-engineering advanced rapidly in the last decade and motivated numerous studies in cell-engineering and biofabrication. Three-dimensional (3D) tissue-engineering scaffolds play a critical role in this field, as the scaffolds provide the biomimetic microenvironments that could stimulate desired cell behaviors for regeneration. However, despite many achievements, the fabrication of 3D scaffold remains challenging due to the difficulty of encapsulating cells in 3D scaffolds, controlling cell-cell organization in 3D, and being adapted by users unfamiliar with 3D biofabrication. In this study, we circumvent these obstacles by creating a four-dimensional (4D) inkjet-printing platform. This platform produces micropatterns that self-fold into a 3D scaffold. Seeding live cells uniformly onto the micropatterns before self-folding leads to cell-encapsulating 3D scaffolds with layer-wise cell-cell organization. Photo-crosslinkable biomaterial-inks of distinct swelling rates were synthesized from gelatin, and the biomaterial-inks were patterned by a customized high-precision inkjet-printer into bilayer micropatterns that were capable of self-folding into 3D microstructures. A mathematical model was developed to help design self-folding and to aid the understanding of the self-folding mechanism. Human umbilical vein endothelial cells (HUVECs) were embedded in self-folded microtubes to mimic microvessels. HUVECs in the microtube spread, proliferated, showed high cell viability, and engrafted on the microtube's inner wall mimicking the native endothelial cells. For physician and biologist end-users, this 4D printing method provides an easy-to-use platform that supports standard two-dimensional cell-seeding protocol while enabling the users to customize 3D cellularized scaffold as desired. This work demonstrated 4D printing as a promising tool for tissue-engineering applications.

4D biofabrication via instantly generated graded hydrogel scaffolds

“…Compared with 3D scaffolds, 4D scaffolds with the ability to reconfigure their shapes during culture show huge potential for morphodynamic tissue engineering. Hydrogels that harness non-uniform swelling [ 43 , 54 , [56] , [57] , [58] ], post-programmed anisotropic internal strains [ 59 , 60 ], or cell contractile forces [ [61] , [62] , [63] ] can accomplish this task. However, in addition to the stringent requirements regarding material cytocompatibility, the fabrication process, and imposed stimulation, complexity in fabrication and lack of controllability present a significant impedance to 4D tissue engineering.…”

Four-Dimensional Printing Technology at the Frontier of Advanced Modeling and Applications in Brain Tissue Engineering

“…Gellan gum-RGD (RGD-GG) [57] CNS grafts [58] 10% GelMA and 15% PEGDA [63] S 4-arm PLA [64] SOEA [61] AlgGel [62] Printing Technique…”

Natural Origin Biomaterials for 4D Bioprinting Tissue‐Like Constructs

“…One limitation of the bioprinting techniques in general, whether it is 3D or 4D, is that the physical entrapment of cells within the bioprinted constructs may hinder some cellular process, namely spreading, migration, and organization, which may compromise the overall therapeutic performance of the constructs. [60] Controlling cell distribution within the bioprinted construct in the long term is also a very challenging aspect of 3D/4D bioprinting, since the cellular processes taking place post printing may alter the cell distribution within the material, which may result in inhomogeneous cell distribution and even the formation of cell clusters within the bioprinted material. [125] Many of the studies carried out on 4D-bioprinted constructs focus a lot more on the shape or functionality change process of the final material and end up lacking more comprehensive studies on how these changes affect complex cellular processes, oftentimes only performing biological assays based on staining techniques (e.g., live/dead viability assay).…”

“…[16] This strategy can also be applied to achieve other desirable properties, [16] such as solubility in water, [54] control over the degradation rate, [55] and enhanced mechanical properties. [56] Gathering on this, some of the most explored natural polymers for 4D bioprinting including alginate, [33,57] collagen, [58] gelatin, [34,59,60] hyaluronic acid, [18,33] and chitosan [61] are depicted in Figure 1. Within 3D bioprinting applications, alginate hydrogel bioinks are undoubtedly one of the most broadly researched natural biomaterials.…”

“…In a more recent study, Cui et al developed a new approach to produce shape morphing 4D-printed hydrogels from gelatin-based bioinks (GelMA and GelCOOHMA). [60] In this approach, the bioprinting of the shape morphing hydrogel is performed on a glass slide coated with a sacrificial layer of alginate/gelatin, to allow detachment and folding of this structure in due time, after umbilical vein endothelial cells are properly seeded and cultured on its surface. After detaching, the different swelling rate of GelMA and GelCOOHMA in aqueous solutions causes the hydrogels to self-fold.…”