Project Summary Restoration of soft tissue is a significant challenge facing clinicians. The available reparative options, whether prosthetic or autologous, present major drawbacks including donor site defects, unpredictable tissue survival, limited duration of restoration, prosthetic exposure, infection, and fibrosis. Therefore, a critical need exists for a solution which can replace missing tissue volume while encouraging natural remodeling of soft tissue over time. We have recently developed an injectable nanofiber-hydrogel composite (NHC) material which is capable of inducing remodeling of the injected volume into vascularized soft tissue with adipocytes without relying on exogenous growth factors and cells. However, the mechanisms by which it does so are not yet well understood. The goal of this proposed project is to characterize the immune and tissue remodeling kinetics of the injected NHC material, uncover the mechanism(s) by which it achieves soft tissue remodeling, and optimize its formulation to enhance this desired outcome. We will first investigate the local immune and tissue remodeling kinetics of the injected site caused by the NHC. This will include characterizing cell infiltration, healthy extracellular matrix (ECM) deposition vs. irregular fibrosis and foreign body response, inflammation, angiogenesis, and adipogenesis. In addition, we will uncover the NHC’s mechanism of instigating soft tissue remodeling by carrying out single-cell RNA sequencing to identify candidate cell subtypes and activated signaling pathways and then subsequently confirm them through immune knockout or targeted depletion models to establish a causal relationship. Gaining insight into how soft tissue remodeling can be accomplished with this biomaterial system will have wide-reaching implications for the regenerative medicine field as we work towards creating off-the-shelf biomaterials-based solutions for tissue replacement. Finally, through modulation of physical and structural properties as well as leveraging our mechanistic understanding, we will optimize the parameters of the NHC to maximize desired soft tissue remodeling. If successful, this proposal will produce an improved off- the-shelf biostimulatory NHC with enhanced capacity to achieve natural tissue restoration outcomes as well as a mechanistic understanding of how to achieve soft tissue remodeling, thereby expanding the ability to treat patients and allowing scientists to engineer the next generation of biomaterials for tissue replacement.