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Bulk hydrogels traditionally used for tissue engineering and drug delivery have numerous limitations, such as restricted injectability and a nanoscale porosity that reduces cell invasion and mass transport. An evolving approach to address these limitations is the fabrication of hydrogel microparticles (i.e., "microgels") that can be assembled into granular hydrogels. There are numerous methods to fabricate microgels; however, the influence of the fabrication technique on granular hydrogel properties is unexplored. Herein, we investigated the influence of three microgel fabrication techniques (microfluidic devices (MD), batch emulsions (BE), and mechanical fragmentation by extrusion (EF)) on the resulting granular hydrogel properties (e.g., mechanics, porosity, and injectability). Hyaluronic acid (HA) modified with various reactive groups (i.e., norbornenes (NorHA), pentenoates (HA-PA), and methacrylates (MeHA)) were used to form microgels with an average diameter of ∼100 μm. The MD method resulted in homogeneous spherical microgels, the BE method resulted in heterogeneous spherical microgels, and the EF method resulted in heterogeneous polygonal microgels. Across the various reactive groups, microgels fabricated with the MD and BE methods had lower functional group consumption when compared to microgels fabricated with the EF method. When microgels were jammed into granular hydrogels, the storage modulus (G') of EF granular hydrogels (∼1000-3000 Pa) was consistently an order of magnitude higher than G' for MD and BE granular hydrogels (∼50-200 Pa). Void space was comparable across all groups, although EF granular hydrogels exhibited an increased number of pores and decreased average pore size when compared to MD and BE granular hydrogels. Furthermore, granular hydrogel properties were tuned by varying the amount of cross-linker used during microgel fabrication. Lastly, granular hydrogels were injectable across formulations due to their general shear-thinning and self-healing properties. Taken together, this work thoroughly characterizes the influence of the microgel fabrication technique on granular hydrogel properties to inform the design of future systems for biomedical applications.

Biopolymers are natural polymers sourced from plants and animals, which include a variety of polysaccharides and polypeptides. The inclusion of biopolymers into biomedical hydrogels is of great interest because of their inherent biochemical and biophysical properties, such as cellular adhesion, degradation, and viscoelasticity. The objective of this Review is to provide a detailed overview of the design and development of biopolymer hydrogels for biomedical applications, with an emphasis on biopolymer chemical modifications and cross-linking methods. First, the fundamentals of biopolymers and chemical conjugation methods to introduce cross-linking groups are described. Cross-linking methods to form biopolymer networks are then discussed in detail, including (i) covalent cross-linking (e.g., free radical chain polymerization, click cross-linking, cross-linking due to oxidation of phenolic groups), (ii) dynamic covalent cross-linking (e.g., Schiff base formation, disulfide formation, reversible Diels-Alder reactions), and (iii) physical cross-linking (e.g., guest-host interactions, hydrogen bonding, metal-ligand coordination, grafted biopolymers). Finally, recent advances in the use of chemically modified biopolymer hydrogels for the biofabrication of tissue scaffolds, therapeutic delivery, tissue adhesives and sealants, as well as the formation of interpenetrating network biopolymer hydrogels, are highlighted.

Granular hydrogels are formed through the packing of hydrogel microparticles and are emerging for various biomedical applications, including as inks for 3D printing, substrates to study cell-matrix interactions, and injectable scaffolds for tissue repair. Granular hydrogels are suited for these applications because of their unique properties including inherent porosity, shear-thinning and self-healing behavior, and tunable design. The characterization of their material properties and biological response involves technical considerations that are unique to modular systems like granular hydrogels. Here, we describe detailed methods that can be used to quantitatively characterize the rheological behavior and porosity of granular hydrogels using reagents, tools, and equipment that are typically available in biomedical engineering laboratories. In addition, we detail methods for 3D cell invasion assays using multicellular spheroids embedded within granular hydrogels and describe steps to quantify features of cell outgrowth (e.g., endothelial cell sprouting) using standard image processing software. To illustrate these methods, we provide examples where features of granular hydrogels such as the size of hydrogel microparticles and their extent of packing during granular hydrogel formation are modulated. Our intent with this resource is to increase accessibility to granular hydrogel technology and to facilitate the investigation of granular hydrogels for biomedical applications.

Granular hydrogels are an exciting class of microporous and injectable biomaterials that are being explored for many biomedical applications, including regenerative medicine, 3D printing, and drug delivery. Granular hydrogels often possess low mechanical moduli and lack structural integrity due to weak physical interactions between microgels. This has been addressed through covalent inter-particle crosslinking; however, covalent crosslinking often occurs through temporal enzymatic methods or photoinitiated reactions, which may limit injectability and material processing. To address this, a hyaluronic acid (HA) granular hydrogel is developed with dynamic covalent (hydrazone) inter-particle crosslinks. Extrusion fragmentation is used to fabricate microgels from photocrosslinkable norbornene-modified HA, additionally modified with either aldehyde or hydrazide groups. Aldehyde and hydrazide-containing microgels are mixed and jammed to form adhesive granular hydrogels. These granular hydrogels possess enhanced mechanical integrity and shape stability over controls due to the covalent inter-particle bonds, while maintaining injectability due to the dynamic hydrazone bonds. The adhesive granular hydrogels are applied to 3D printing, which allows the printing of structures that are stable without any further post-processing. Additionally, the authors demonstrate that adhesive granular hydrogels allow for cell invasion in vitro. Overall, this work demonstrates the use of dynamic covalent inter-particle crosslinking to enhance injectable granular hydrogels.

Granular hydrogels have emerged as a new class of injectable and porous biomaterials that improve integration with host tissue when compared to solid hydrogels. Granular hydrogels are typically prepared using spherical particles and this study considers whether particle shape (i.e., isotropic spheres versus anisotropic rods) influences granular hydrogel properties and cellular invasion. Simulations predict that anisotropic rods influence pore shape and interconnectivity, as well as bead transport through granular assemblies. Photocrosslinkable norbornene-modified hyaluronic acid is used to produce spherical and rod-shaped particles using microfluidic droplet generators and formed into shear-thinning and self-healing granular hydrogels at low and high particle packing. Rod-shaped particles form granular hydrogels that have anisotropic and interconnected pores, with pore number and size, storage moduli, and extrusion forces influenced by particle shape and packing. Robust in vitro sprouting of endothelial cells from embedded cellular spheroids is observed with rod-shaped particles, including higher sprouting densities and sprout lengths when compared to hydrogels with spherical particles. Cellular invasion into granular hydrogels when injected subcutaneously in vivo is significantly greater with rod-shaped particles, whereas a gradient of cellularity is observed with spherical particles. Overall, this work demonstrates potentially superior functional properties of granular hydrogels with rod-shaped particles for tissue repair.

Two of the most prominent challenges that limit the clinical success of siRNA therapies are a lack of control over cargo release from the delivery vehicle and an incomplete understanding of the link between gene silencing dynamics and siRNA dosing. Herein, we address these challenges through the formulation of siRNA polyplexes containing light-responsive polymer mixtures, whose varied compositions and triggered release behavior provide enhanced gene silencing and controlled dose responses that can be predicted by simple kinetic models. Through the straightforward mixing of two block copolymers, the level of gene knockdown was easily optimized to achieve the maximum level of GAPDH protein silencing in NIH/3T3 cells (~70%) using a single siRNA dose. The kinetic model was used to describe the dynamic changes in mRNA and protein concentrations in response to siRNA treatment. These predictions enabled the application of a second dose of siRNA to maximally suppress gene expression over multiple days, leading to a further 50% reduction in protein levels relative to those measured following a single dose. Furthermore, polyplexes remained dormant in cells until exposed to the photo-stimulus, demonstrating the complete control over siRNA activity as well as the stability of the nanocarriers. Thus, this work demonstrates that pairing advances in biomaterials design with simple kinetic modeling provides new insight into gene silencing dynamics and presents a powerful strategy to control gene expression through siRNA delivery. Statement of Significance Our manuscript describes two noteworthy impacts: (1) we designed mixed polymer formulations to enhance gene silencing, and (2) we simultaneously developed a simple kinetic model for determining optimal siRNA dose responses to maintain silencing over several days. These advances address critical challenges in siRNA delivery and provide new opportunities in therapeutics development. The structure-function relationships prevalent in these formulations were established to enable tuning and forecasting of nanocarrier efficiency a priori , leading to siRNA dosing regimens able to maximally suppress gene expression. Our advances are significant because the mixed polymer formulations provide a straightforward and scalable approach to tailor siRNA delivery regimens. Moreover, the implementation of accurate dosing frameworks addresses a major knowledge gap that has hindered clinical implementation of siRNA.