Project Summary The regeneration of damaged or diseased tissues that serve biomechanical functions, such as musculoskeletal tissues, has been a long-standing challenge in clinical practice and research. Regenerative engineering offers a promising alternative to auto- or allografts for tissue regeneration by combining biomaterial scaffolds, viable cells, and bioactive factors. Engineering scaffolds that provide both mechanical support and biological activities is critical for regenerating such tissues with biomechanical functions. However, currently existing scaffolds, which include either tough polymers with limited bioactivities or soft hydrogels with poor mechanical properties, fall short of meeting both mechanical and biological needs. To address this issue, we propose the development of a novel family of emulsion bioinks to enable the 3D bioprinting of strong living scaffolds with built-in mechanical robustness and desirable biological functions for tissue regeneration. The encapsulation of biologics (cells and bioactive factors) within scaffolds presents an attractive strategy to equip the scaffolds with desired biological functions. The major roadblocks to encapsulate biologics within tough polymers include their lack of bioactivity and the frequent usage of harmful chemicals, such as organic solvents and/or toxic reactants. In this study, a water-in-oil emulsion bioink is designed by dispersing an aqueous internal phase of hydrogel droplets (microgels) with encapsulated biologics in an external phase of tough polymer solution. It is hypothesized that microgels will protect the functions of encapsulated biologics from harmful chemicals by limiting their diffusion from the external to internal phases. The solidification of tough polymer around each dispersed microgel during 3D-bioprinting will mainly contribute to mechanical robustness of the final scaffold. The preliminary data demonstrates that: 1) >95% viability of fibroblast cells is achieved in an emulsion bioink; and 2) the resulting emulsion scaffolds afford both the mechanical robustness (elastic moduli 5-40 MPa) and >90% cell viability. This project will initiate with the development of cytocompatible and bioprintable cell- laden emulsion bioinks, followed by characterization of 3D-bioprinted emulsion scaffolds, and conclude with validating the functions of encapsulated bioactive factors and cells within scaffolds for meniscus regeneration as a test model. This model will include assessments of proliferation, fibrochondrogenic differentiation in vitro, and neo-menisci formation in vivo. Overall, our approach presents a new method to produce mechanically strong and biologically functional living scaffolds by integrating emulsion chemistry and 3D bioprinting technology. We anticipate that this work will have a broad and significant impact on regenerative engineering by benefiting repair or regeneration of broad-spectrum tissues with biomechanical functions.