From Soft Microgel Assemblies to Advanced Healthcare Materials

In 2015, my laboratory (seguralab.duke.edu), in collaboration with Dino Di Carlo's lab, published the use of injectable soft microgel assemblies as scaffolds to promote fast stem cell expansion and endogenous tissue repair.[1] What has followed is a quick adaption of our technology to many applications in biomaterials science, including cell expansion, cell delivery, endogenous tissue repair, and 3D printing. This Special issue in Advanced Healthcare Materials aims to capture current applications in injectable soft microgel assemblies for biomedical applications, including microporous annealed particle (MAP) scaffolds, microgel engineering, bioactive signal sequestration/delivery, in vitro cell differentiation, endogenous tissue repair, and 3D printing. Our interest in creating an injectable, yet porous hydrogel that did not require degradation of components to generate micron sized pores, necessitated the use of micron size building blocks as opposed to the polymer macromers used to form traditional hydrogels. By increasing the size of the building block to tens-hundreds of microns, we were able to maintain injectability through standard syringes while creating spaces between particles that were large enough for cell growth or infiltration without the need for hydrogel degradation. At the microgel interface, secondary crosslinks generated from an annealable component incorporated during injection or reactive groups at the microgel surface, resulted in stabilized yet flowable scaffolds. This decoupling of material degradation, hydrogel porosity, and cell expansion/tissue infiltration, significantly improved outcomes both in vitro and in vivo, allowing unprecedented rapid cell expansion of mesenchymal stem cells (MSCs) in vitro[1] and improved skin wound closure rate and regeneration of higher order structures.[1] We termed these materials MAP scaffolds. The properties that make MAP scaffolds unique are injectability, microscale porosity, ability to embed cells during the scaffold generation processes, and heterogeneity. These properties have been complemented with new chemical approaches to increase microgel functionality and applications in vitro to understand the role of confinement on cell phenotype, 3D printing to create sophisticated and cell-friendly structures, and in vivo for endogenous tissue repair. Prior to our introduction to MAP scaffolds, soft microgel assemblies for cell culture relied on solvent-solvent extraction of poly(ethylene glycol) and dextran to generate a dense microgel particle assemblies that formed stable scaffolds.[2-4] These microgel assemblies had many of the same features of MAP scaffolds, porosity and biocompatibility, except for some key differences the most notable of which is the lack of injectability, which is critical to the applications showcased in this special issue. Injectability allows for minimally invasive in vivo applications and 3D printing of user defined structures. Further, by generating microgels with defined size and injecting/pipetting them without over packing (deformation along the particle boundaries), the void structure is completely interconnected and open, removing the need for porogen incorporation, allowing for rapid cell expansion and cellular infiltration. The introduction of a material into the body necessitates the engagement of the immune system. This response to a foreign material can be engineered to either lead to pro-fibrotic/inflammatory responses or non-fibrotic/pro-regenerative responses. In this topic we have a review by Edward Phelps and colleagues (202303005), who give a very thorough analysis for the literature to date on the role of microparticle assembly characteristics (e.g., size, stiffness, adhesivity, time). It also compares granular and non-granular hydrogels and demonstrates that granular hydrogels have superior early biocompatibility due to rapid cellular infiltration (innate immune response) and long-term outcomes (adaptive immune response) due to both porosity and bioactivity. We also have several primary research publications that take advantage of MAPs biocompatibility and injectability to deliver islet cell transplantation in a diabetic model (202301552), promote at type 2 immune response through IL-33 conjugation to MAP (202400249), and use two-species MAP to sequester inflammatory cytokine IL-6 with one microgel species, while delivering VEGF from the second microgel species (202400800). Because of MAPs and other soft granular scaffold's superior biocompatibility, they have found applications in promoting endogenous repair in hard-to-treat tissues such as spinal cord injury and brain wounds. This issue of Advanced Healthcare Materials has two primary research articles using MAP to promote spinal cord injury and two focusing on brain healing. Given the delicate nature of both tissues and the current lack of effective treatments for injuries to the central nervous system, these research articles are particularly exciting. Using MAP gels produced from different size microgels, it was found that 60 µm diameter particles significantly improve locomotion, axonal regeneration, remyelination (202302498). Further, MAP was used to deliver neural progenitor cells in a spinal cord injury model and showed enhanced engraftment, and differentiation toward neuronal and astrocytic lineages (202303912). Traumatic brain injury was treated with core/shell microgels (MINOR) containing nitric oxide in the core to promote angiogenesis and dopamine in the shell to work as a ROS scavenger (202302315). MINOR reduces inflammation, increases angiogenesis, and results in functional improvement. Finally, in a model of stroke, MAP generated from cyogels (porous microgels) or regular microgels showed that cryoMAP increased angiogenesis over regular microgels. Further, SDF1 delivery from cryoMAP showed enhanced progenitor cell infiltration and angiogenesis into the stroke core (202302081). Soft microgel assemblies have found increased applications in vitro to study cell behaviors in these defined environments. First, mixing fibroblasts and microgels without adding microgel interlinking agent, results in microgel-cell-microgel annealing with unique shapes emerging as the cells expand that are dependent on the cell density used (202302957). Further, PEG-lipoic acid microgels are formed by inverse suspension polymerization, photodeformed and used to show that C2C12s localize to areas of high curvature (202302925). Last, microgels were synthesized with PEDOT:PSS to introduce varying conductivity, but keeping mechanical properties constant. In these MAP scaffolds void volume fraction and conductivity influence myogenic differentiation in C2C12s and human myoblasts (202302500). The void space of MAP scaffolds can be divided into natural pockets of open space, 3D pores. The size of these pores can be changed through changing the diameter of the microgel,[5-7] and through changing the packing fraction of the microgels through extended gravitational sedimentation,[8] through lyophilization and rehydration in user defined volumes,[9] through vacuum filtration,[10] and through centrifugation. Because hydrogel microparticles are soft and thus can deform at the interface decreasing the void space, this feature can be further exploited to change the shape of the 3D pores. Pore size and shape change MAP scaffold mechanical properties and guide and direct cell phenotype, underscoring the importance of engineering the void space of MAP scaffolds. Previously, it was found that the particle fraction of MAP scaffolds is a bioactive cue for cells, with spreading and proliferation changing as a function of particle packing fraction.[9] We have an example of this presented in this issue, where pore microarchitecture in granular hydrogels was engineered using controlled centrifugation to tailor pore size and shape to direct cell and tissue responses (202402489). So far, the articles described focus on spherical soft microgels assemblies, however, there is increased interest non-spherical particles because they can provide with a different microstructure, 3D pore shape, mechanical properties, and functionality. In this topic, we have a review that presents the methods currently used to generate non-spherical particles and rationale for using non-spherical particles (202301597) and a primary research article reporting, for the first time, on crescent shaped uniform microgels (202302477). These microgels were produced using microfluidic approaches with gelatin as a sacrificial component to generate the crescent shape. They found that MAP scaffold generated from crescent shaped particles resulted in increased infiltration of myofibroblasts and leukocytes. Another method to make microgels is through fragmentation. In this approach a traditional hydrogel is generated and subsequently fragmented into small microgels. The advantages of this type of hydrogel is the ease of production and the ability to load many different types of cargo. We have three primary research articles in this special issue covering the introduction of ZrO2 nanoparticles to allow radiopacity, which allows visualization of gel during injection and in post-injection monitoring. (202303576). Further, the role of microgel size on pore size and rheological properties is investigated (202303326). Last, fragmented microgels and subsequent annealing into a scaffold were generated using zwitterionic, which introduce exciting new properties to granular hydrogels of nonfouling, very high hydrophilicity, and low immunogenicity (202301831). The last topic presented in this special issue is 3D printing. Soft microgel assemblies have found unique applications in 3D printing including as printing baths and as the ink. This topic we have a review article that discusses current advance in the use of soft microgel assemblies for 3D printing including properties of the microgels that influences the use of these particles as inks or printing baths (202301388). We also have two primary research articles publications, one on a new ink for 3D printing and the other on microgel support baths. To improve the fidelity with increased cell viability a biphasic is introduced that allows for jamming without sacrificing porosity (202303810). Last, we have a primary research publication that uses a granular hydrogel bath to print MSCs. They find that printing MSCs into this bath leads to spreading while printing to non-granular baths does not (202303325). Soft microgel assemblies, including MAP scaffolds, belong to the larger field of granular materials. Granular materials are composed of particle units that together jam to form a solid structure. One of the most common granular materials is sand. Individual grains cannot support the weight of a person, but collectively sand can jam and support you walking down the beach. The compactness of the sand influences how much water gets released when you walk in wet sand. To better understand how the open space in granular materials drives function including biological changes such as cell phenotype, strategies to segment the void space of granular materials into local pockets of space is needed. My lab has begun to explore computational approaches to characterize the 3D pores of microparticle assemblies.[11] Further approaches will be needed to understand how the 3D pores and overall void space evolves as cells proliferate, cells infiltrate, and microgels are degraded as well as correlate the evolving microstructural changes with biological phenomena. It is an exciting time for the field of soft microgel assemblies for biomedical applications. The authors declare no conflict of interest. Tatiana Segura is a Professor at Duke University, where she has split appointments between Pratt School of Engineering (Biomedical Engineering) and the School of Medicine (Neurology and Dermatology). Professor Segura's Laboratory studies the design and use of materials to help our body heal itself. In this topic she has published over 100 manuscripts with over 12 000 citations. She has been recognized with the 2024 Clemson Award for Contributions to the Literature from the Society for Biomaterials, the 2020 Acta Biomaterialia Silver Medal Award, 2009 Outstanding Young Investigator Award from the American Society of Gene and Cell Therapy, the American Heart Association National Scientist Development Grant, and the CAREER award from National Science Foundation and has been Elected to the College of Fellows at the American Institute for Medical and Biological Engineers (AIMBE) and as a Senior Member of the National Academy of Inventors. She is currently the director of the Center for Biomolecular and Tissue Engineering at Duke University.