Bone healing involves sequential and overlapping biological processes including inflammation, new bone formation and remodeling. Most bone tissue engineering research to date focused on targeting bone-forming cells such as stem cells. Emerging studies highlight the critical roles of immune cells in fracture healing. Macrophages (M𝜙) is one of the first responders to bone defects and can be polarized into pro-inflammatory M1 and pro-regenerative M2 phenotype. Unsolved acute inflammatory phase and delayed M1 to M2 phenotype switch often lead to long-term and chronic inflammation, resulting in delayed bone healing. To enhance bone healing, there remains a lack of strategy that promotes desirable M𝜙 polarization at appropriate timing. Biomaterial scaffolds have been widely used for bone tissue engineering as carriers for cell and growth factors delivery. Recent studies also demonstrate biomaterials compositions impact immune responses, with natural extracellular derived materials promote more pro-regenerative immune response than synthetic materials. However, several key gaps in knowledge remain. First, previous studies were done using soft-tissue defect models only, and how varying biomaterials compositions impact bone healing remains unknown. Second, macroporosity is critical for bone regeneration in vivo, whereas previous work on assessing immune response to biomaterials is limited to conventional nanoporous hydrogels. Third, previous work on the role of T cells in bone healing is limited to a non-critical size long bone fracture model, and no scaffolds were used. T-cell response to scaffold implant in a critical-sized cranial defect model has never been studied before. Last, previous work only studied individual cell type (i.e. immune cell only or stem cell only), yet how biomaterials composition modulates cell-cell crosstalk and subsequent tissue regeneration remains largely unknown. The goal of our original R01 was to assess the potential of µRB scaffolds for enhancing stem cell-based tissue regeneration by focusing on stem cell differentiation. The goal of this renewal R01 application is to harness µRB scaffolds to enhance bone formation through immunomodulation by tuning biomaterial composition, which has never been investigated before. Specifically, we propose to (1) Assess the effect of varying µRB scaffold composition on M𝜙 polarization and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro; (2) Investigate the effect of the varying µRB composition on MSC/M𝜙 crosstalk, and further impacts on MSC-based bone formation and T cell response using a 3D co-culture model in vitro; and (3) Evaluate the effect of the varying µRB scaffold composition on immune responses and bone regeneration in vivo using a mouse critical-size cranial defect model. By working at the interface of biomaterials, immunology, bone disease and biology, stem cells, animal models, and high dimensional single cell analyses, the proposed work will fill in the...