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Abstract BackgroundEngineered vasculature is essential for the biofabrication of functional tissue mimics. To fabricate engineered vasculature, three-dimensional (3D) bioprinting has emerged as a promising approach due to its ability to form perfusable structures with customized geometries. Sacrificial ink extrusion, where sacrificial inks are printed into a crosslinkable hydrogel precursor support bath, is a versatile bioprinting modality for fabricating interconnected perfusable networks. However, the fabrication of self-supporting structures with a vessel-like shell remains challenging using conventional sacrificial ink extrusion approaches. To enable the fabrication of self-supporting, perfusable networks, we developed a 3D bioprinting approach termed Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE-3DP). This approach leverages the diffusion of crosslinking initiators from a printed sacrificial ink into a gel precursor support bath to generate branched, perfusable networks with precise control over channel inner and outer diameters. MethodsHere, we present an end-to-end protocol for fabricating self-supporting vascular-like networks using the GUIDE-3DP method. We describe methods for freeform print path design, support bath and sacrificial ink preparation, 3D printing of perfusable structures, and seeding of printed structures with endothelial cells. Through this protocol, perfusable structures with complex branching geometries can be designed, fabricated, and endothelialized. DiscussionTo highlight the ability of GUIDE-3DP to fabricate self-supporting, perfusable networks with complex geometries, we demonstrate the fabrication of three representative structures: (1) an interconnected retinal vasculature network, (2) a hierarchical branched vascular network, and (3) a dual-material capillary-like network. We further demonstrate the endothelialization of printed structures with one or two cell types via single- or dual-material printing. Beyond vascular-like networks, this protocol is readily adaptable to design and fabricate mimics of other perfusable structures in the human body. Clinical trial numberNot applicable. 

Abstract The development of three-dimensional (3D)in vitrotissue culture models is critical for biomedical research. Hydrogel-based systems have become a preferred scaffold for 3D models, as they have tunable viscoelastic properties, which are well-known to influence cell morphology and function. In particular, reversible hydrogel crosslinks formed through dynamic covalent chemistry (DCC) can introduce viscoelastic behavior including stress relaxation. However, traditional strategies to increase stress-relaxation rates in DCC gels rely on faster bond kinetics, resulting in faster erosion rates that prevent their use for long-term 3D culture. As an alternative strategy, we explore the use of molecular parameters (specifically molecular weight and degree of functionalization) to independently control the stiffness and stress relaxation behavior while preventing rapid erosion. As demonstration, we develop and validate a modified theoretical model of gel viscoelasticity applied to a two-component DCC gel composed of modified hyaluronic acid and elastin-like protein. Finally, we utilize this tunable gel platform to explore the impact of scaffold viscoelasticity on encapsulated human neural progenitor cells. In summary, this work expands the molecular design space of DCC hydrogels to achieve tunable viscoelastic properties for 3Din vitromodels. 

Abstract The biochemical and biophysical properties of the extracellular matrix (ECM) play a pivotal role in regulating cellular behaviors such as proliferation, migration, and differentiation. Engineered protein‐based hydrogels, with highly tunable multifunctional properties, have the potential to replicate key features of the native ECM. Formed by self‐assembly or crosslinking, engineered protein‐based hydrogels can induce a range of cell behaviors through bioactive and functional domains incorporated into the polymer backbone. Using recombinant techniques, the amino acid sequence of the protein backbone can be designed with precise control over the chain‐length, folded structure, and cell‐interaction sites. In this review, the modular design of engineered protein‐based hydrogels from both a molecular‐ and network‐level perspective are discussed, and summarize recent progress and case studies to highlight the diverse strategies used to construct biomimetic scaffolds. This review focuses on amino acid sequences that form structural blocks, bioactive blocks, and stimuli‐responsive blocks designed into the protein backbone for highly precise and tunable control of scaffold properties. Both physical and chemical methods to stabilize dynamic protein networks with defined structure and bioactivity for cell culture applications are discussed. Finally, a discussion of future directions of engineered protein‐based hydrogels as biomimetic cellular scaffolds is concluded. 

Neural progenitor cells (NPCs) hold immense potential as therapeutic candidates for neural regeneration, and materials-based strategies have emerged as attractive options for NPC expansion. However, maintaining NPC stemness has proven challenging in vitro, due to their propensity to form cell-dense neurospheres. While neurospheres promote cell-cell interactions required for NPC stem maintenance, they also restrict oxygen transport, leading to hypoxia and limited cell expansion. To overcome these limitations, we investigate two materials-based approaches to maintain NPC stemness: 1) physical matrix remodeling within a viscoelastic, stress-relaxing hydrogel and 2) matrix-induced N-cadherin-like signaling through a cell-instructive peptide. While viscoelasticity alone is sufficient to maintain NPC stemness compared to an elastic environment, NPCs still preferentially form neurospheres. The addition of N-cadherin-like peptides promotes a distributed culture of NPCs while maintaining their stemness through cadherin-mediated signaling, ultimately exhibiting improved long-term expansion and neural differentiation. Thus, our findings reveal matrix viscoelasticity and engineered N-cadherin-like interactions as having a synergistic effect on NPC expansion and differentiation within 3D matrices.