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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. 

Abstract 3D bioprinting has enabled the fabrication of tissue‐mimetic constructs with freeform designs that include living cells. In the development of new bioprinting techniques, the controlled use of diffusion has become an emerging strategy to tailor the properties and geometry of printed constructs. Specifically, the diffusion of molecules with specialized functions, including crosslinkers, catalysts, growth factors, or viscosity‐modulating agents, across the interface of printed constructs will directly affect material properties such as microstructure, stiffness, and biochemistry, all of which can impact cell phenotype. For example, diffusion‐induced gelation is employed to generate constructs with multiple materials, dynamic mechanical properties, and perfusable geometries. In general, these diffusion‐based bioprinting strategies can be categorized into those based on inward diffusion (i.e., into the printed ink from the surrounding air, solution, or support bath), outward diffusion (i.e., from the printed ink into the surroundings), or diffusion within the printed construct (i.e., from one zone to another). This review provides an overview of recent advances in diffusion‐based bioprinting strategies, discusses emerging methods to characterize and predict diffusion in bioprinting, and highlights promising next steps in applying diffusion‐based strategies to overcome current limitations in biofabrication. 

Abstract Dynamic covalent chemistry (DCC) crosslinks can form hydrogels with tunable mechanical properties permissive to injectability and self‐healing. However, not all hydrogels with transient crosslinks are easily extrudable. For this reason, two additional design parameters must be considered when formulating DCC‐crosslinked hydrogels: 1) degree of functionalization (DoF) and 2) polymer molecular weight (MW). To investigate these parameters, hydrogels comprised of two recombinant biopolymers: 1) a hyaluronic acid (HA) modified with benzaldehyde and 2) an elastin‐like protein (ELP) modified with hydrazine (ELP‐HYD), are formulated. Several hydrogel families are synthesized with distinct HA MW and DoF while keeping the ELP‐HYD component constant. The resulting hydrogels have a range of stiffnesses,G′ ≈ 10–1000 Pa, and extrudability, which is attributed to the combined effects of DCC crosslinks and polymer entanglements. In general, lower MW formulations require lower forces for injectability, regardless of stiffness. Higher DoF formulations exhibit more rapid self‐healing. Gel extrusion through a cannula (2 m length, 0.25 mm diameter) demonstrates the potential for minimally invasive delivery for future biomedical applications. In summary, this work highlights additional parameters that influence the injectability and network formation of DCC‐crosslinked hydrogels and aims to guide future design of injectable hydrogels. 

Abstract Microextrusion‐based 3D bioprinting into support baths has emerged as a promising technique to pattern soft biomaterials into complex, macroscopic structures. It is hypothesized that interactions between inks and support baths, which are often composed of granular microgels, can be modulated to control the microscopic structure within these macroscopic‐printed constructs. Using printed collagen bioinks crosslinked either through physical self‐assembly or bioorthogonal covalent chemistry, it is demonstrated that microscopic porosity is introduced into collagen inks printed into microgel support baths but not bulk gel support baths. The overall porosity is governed by the ratio between the ink's shear viscosity and the microgel support bath's zero‐shear viscosity. By adjusting the flow rate during extrusion, the ink's shear viscosity is modulated, thus controlling the extent of microscopic porosity independent of the ink composition. For covalently crosslinked collagen, printing into support baths comprised of gelatin microgels (15‐50 µm) results in large pores (≈40 µm) that allow human corneal mesenchymal stromal cells (MSCs) to readily spread, while control samples of cast collagen or collagen printed in non‐granular support baths do not allow cell spreading. Taken together, these data demonstrate a new method to impart controlled microscale porosity into 3D printed hydrogels using granular microgel support baths. 

Abstract The encapsulation of cells within gel‐phase materials to form bioinks offers distinct advantages for next‐generation 3D bioprinting. 3D bioprinting has emerged as a promising tool for patterning cells, but the technology remains limited in its ability to produce biofunctional, tissue‐like constructs due to a dearth of materials suitable for bioinks. While early demonstrations commonly used viscous polymers optimized for printability, these materials often lacked cell compatibility and biological functionality. In response, advanced materials that exist in the gel phase during the entire printing process are being developed, since hydrogels are uniquely positioned to both protect cells during extrusion and provide biological signals to embedded cells as the construct matures during culture. Here, an overview of the design considerations for gel‐phase materials as bioinks is presented, with a focus on their mechanical, biochemical, and dynamic gel properties. Current challenges and opportunities that arise due to the fact that bioprinted constructs are active, living hydrogels composed of both acellular and cellular components are also evaluated. Engineering hydrogels with consideration of cells as an intrinsic component of the printed bioink will enable control over the evolution of the living construct after printing to achieve greater biofunctionality. 

Abstract Granular, microgel‐based materials have garnered interest as promising tissue engineering scaffolds due to their inherent porosity, which can promote cell infiltration. Adapting these materials for 3D bioprinting, while maintaining sufficient void space to enable cell migration, can be challenging, since the rheological properties that determine printability are strongly influenced by microgel packing and void fraction. In this work, a strategy is proposed to decouple printability and void fraction by blending UV‐crosslinkable gelatin methacryloyl (GelMA) microgels with sacrificial gelatin microgels to form composite inks. It is observed that inks with an apparent viscosity greater than ≈100 Pa s (corresponding to microgel concentrations ≥5 wt%) have rheological properties that enable extrusion‐based printing of multilayered structures in air. By altering the ratio of GelMA to sacrificial gelatin microgels, while holding total concentration constant at 6 wt%, a family of GelMA:gelatin microgel inks is created that allows for tuning of void fraction from 0.20 to 0.57. Furthermore, human umbilical vein endothelial cells (HUVEC) seeded onto printed constructs are observed to migrate into granular inks in a void fraction‐dependent manner. Thus, the family of microgel inks holds promise for use in 3D printing and tissue engineering applications that rely upon cell infiltration. 

Abstract While the human body has many different examples of perfusable structures with complex geometries, biofabrication methods to replicate this complexity are still lacking. Specifically, the fabrication of self‐supporting, branched networks with multiple channel diameters is particularly challenging. Herein, the Gelation of Uniform Interfacial Diffusant in Embedded 3D Printing (GUIDE‐3DP) approach for constructing perfusable networks of interconnected channels with precise control over branching geometries and vessel sizes is presented. To achieve user‐specified channel dimensions, this technique leverages the predictable diffusion of cross‐linking reaction‐initiators released from sacrificial inks printed within a hydrogel precursor. The versatility of GUIDE‐3DP to be adapted for use with diverse physicochemical cross‐linking mechanisms is demonstrated by designing seven printable material systems. Importantly, GUIDE‐3DP allows for the independent tunability of both the inner and outer diameters of the printed channels and the ability to fabricate seamless junctions at branch points. This 3D bioprinting platform is uniquely suited for fabricating lumenized structures with complex shapes characteristic of multiple hollow vessels throughout the body. As an exemplary application, the fabrication of vasculature‐like networks lined with endothelial cells is demonstrated. GUIDE‐3DP represents an important advance toward the fabrication of self‐supporting, physiologically relevant networks with intricate and perfusable geometries. 

Abstract Mechanically tunable hydrogels are attractive platforms for 3D cell culture, as hydrogel stiffness plays an important role in cell behavior. Traditionally, hydrogel stiffness has been controlled through altering either the polymer concentration or the stoichiometry between crosslinker reactive groups. Here, an alternative strategy based upon tuning the hydrophilicity of an elastin‐like protein (ELP) is presented. ELPs undergo a phase transition that leads to protein aggregation at increasing temperatures. It is hypothesized that increasing this transition temperature through bioconjugation with azide‐containing molecules of increasing hydrophilicity will allow direct control of the resulting gel stiffness by making the crosslinking groups more accessible. These azide‐modified ELPs are crosslinked into hydrogels with bicyclononyne‐modified hyaluronic acid (HA‐BCN) using bioorthogonal, click chemistry, resulting in hydrogels with tunable storage moduli (100–1000 Pa). Human mesenchymal stromal cells (hMSCs), human umbilical vein endothelial cells (HUVECs), and human neural progenitor cells (hNPCs) are all observed to alter their cell morphology when encapsulated within hydrogels of varying stiffness. Taken together, the use of protein hydrophilicity as a lever to tune hydrogel mechanical properties is demonstrated. These hydrogels have tunable moduli over a stiffness range relevant to soft tissues, support the viability of encapsulated cells, and modify cell spreading as a consequence of gel stiffness. 

Abstract Neural progenitor cells (NPCs) are a promising cell source to repair damaged nervous tissue. However, expansion of therapeutically relevant numbers of NPCs and their efficient differentiation into desired mature cell types remains a challenge. Material‐based strategies, including culture within 3D hydrogels, have the potential to overcome these current limitations. An ideal material would enable both NPC expansion and subsequent differentiation within a single platform. It has recently been demonstrated that cell‐mediated remodeling of 3D hydrogels is necessary to maintain the stem cell phenotype of NPCs during expansion, but the role of matrix remodeling on NPC differentiation and maturation remains unknown. By culturing NPCs within engineered protein hydrogels susceptible to degradation by NPC‐secreted proteases, it is identified that a critical amount of remodeling is necessary to enable NPC differentiation, even in highly degradable gels. Chemical induction of differentiation after sufficient remodeling time results in differentiation into astrocytes and neurotransmitter‐responsive neurons. Matrix remodeling modulates expression of the transcriptional co‐activator Yes‐associated protein, which drives expression of NPC stemness factors and maintains NPC differentiation capacity, in a cadherin‐dependent manner. Thus, cell‐remodelable hydrogels are an attractive platform to enable expansion of NPCs followed by differentiation of the cells into mature phenotypes for therapeutic use.