Project Summary Hydrogels are widely used in tissue engineering and regenerative medicine due to their ability to mimic the physical properties of various tissues, host encapsulated cells, and serve as models for diseased and traumatized tissue. Despite these advantages, they face many limitations that hinder their utility and impede their adoption. A primary limitation is their inability to support cell motility and proliferation, the elaboration of extracellular matrix (ECM) components, and the development of de novo tissue. These limitations are primarily attributed to a lack of hierarchical structure bridging the macromolecular and tissue length scales. Conventional, homogeneous polymeric hydrogels, for instance, lack long range architectural features, such as micron-scale porosity. Recently, granular hydrogel scaffolds, a new class of materials composed of densely-packed hydrogel microparticles, has been introduced and rapidly adopted. These scaffolds offer many advantages over conventional hydrogels for tissue engineering. Leveraging advances in high- throughput microfluidic emulsion templating, hydrogel particles can be produced in sufficiently large quantities to enable the assembly of macroscale materials. These granular scaffolds offer significantly increased porosity, facilitating the infiltration of cells and the rapid diffusional exchange of nutrients and waste products. Moreover, particles can be designed independently of macroscale requirements, effectively decoupling material stiffness from porosity. Beyond these design advantages, granular scaffolds can be injected, 3D printed, or molded into arbitrary shapes. This proposal seeks to extend the known advantages of granular gels by incorporating newly developed microfabrication capabilities. The Oakey lab has recently demonstrated the ability to fabricate heterogeneous particles with network architecture sculpted on the nanoscale. This capability offers several unique advantages including tunable rheological properties, controlled degradation, and quantitatively tailored biofunctional interfaces. We will use these capabilities to produce granular scaffolds as a medium to study fundamental and poorly understood questions of cell motility and proliferation within granular scaffolds. This knowledge will then be applied to develop translational applications for granular scaffolds in cartilage regeneration and peripheral nerve allografts. Our interdisciplinary team of researchers have a track record of productive research collaboration and mentoring and each investigator provides a specific and complementary expertise that will inform the rapid acceleration of granular scaffold development. The overlapping aims and activities between all projects will ensure that the findings from each project inform the others. The Specific Aims of this collaborative team project, described as a co-project and led by one investigator, are: co-Project 1: To microfabricate hydrogel particles, cell carriers, and granular scaffolds for each subsequent co-Project. This co-Project will also investigate possible roles for scaffold architecture and particle biochemistry in the assembly of ECM components in de novo tissue. co-Project 2: To develop a scalable experimental platform to visualize and quantitatively assess changes in cell behavior as a response to encapsulation in, with a specific focus upon understanding cell migration and invasion (outgrowth) within 3D granular scaffolds. co-Project 3: To tailor granular scaffolds granular scaffolds to promote chondrocyte outgrowth and thereby cartilage repair by cartilage fragments. co-Project 4: To develop sacrificial granular scaffolds as cell carriers for immunomodulatory regulatory T cell delivery and release to peripheral nerve allografts. The project scope and outcomes connect directly to Wyoming INBRE, an award with research focus areas in cell biology and chronic disease. This supplementary award will use granular gels made by a unique particle fabrication strategy to study questions of fundamental and applied interest to cell biology while informing the technology's translation to address two chronic diseases: functional and traumatic nerve loss and cartilage injury and degradation.

Project Narrative This project aims to understand and develop approaches to tissue repair that utilize granular cell scaffolds, closely packed hydrogel microparticles that emulate tissue structures. This project focuses upon fundamental and applied aspects of tissue repair, including cell motility through granular scaffolds and their application in chronic diseases. This project has the potential to generate new therapeutic strategies for common health issues such as cartilage and nerve damage.