Project Summary/Abstract: Large bone defects are clincially challenging to treat and often necessitate bone grafting. Natural grafting options include autografts and allografts; however, these replacement tissues are limited in supply and difficult to match to the dimensional irregularities of complex bone defects. The development of tissue-engineered (TE) synthetic grafts has become essential to overcome the limitations of natural grafts; however, deficient scaffold fabrication methods and inefficient osteoinductive agents have prevented the clinical translation of traditional TE constructs. Therefore, the design of advanced synthetic grafts that overcome these limitations would greatly impact the clinical treatment of large bone defects. The long-term goal of this proposed work is to develop biodegradable, 3D-printed constructs with osteoconductive and inductive properties toward clinical use for the treatment of patient-specific bone defects. The objective of this proposal aims to develop a TE construct for bone regeneration using a hybrid materials approach that includes both synthetic and natural polymers in the 3D-printed structure, combined with the sustained release of osteoinductive microRNAs. Advanced TE constructs for this investigation will combine 3D-printable, FDA-approved polymers with tunable biodegradation rates with natural polymer coatings to sustain the release of osteoinductive microRNAs. The central hypothesis of this work is that the sustained release of osteoinductive microRNAs from polymer-coated 3D-printed constructs will enhance the osteogenic capabilities of synthetic grafts by prolonging regenerative signaling to maximize bone regeneration. To test this hypothesis, we will characterize microRNA release from polymer- coated 3D-printed constructs (Aim 1), assess in vitro osteogenic differentiation induced by microRNA release from polymer-coated constructs (Aim 2), and evaluate the bone regeneration potential of polymer-coated microRNA-incorporated 3D-printed constructs (Aim 3). Collectively, these data with elucidate mechanisms in which microRNA release from polymer-coated 3D-printed scaffolds can be optimized to sustain the release of osteoinductive signals and maximize bone regeneration. These results will advance the development of synthetic TE constructs to include both osteoconductive and inductive properties that will effectively promote bone regeneration, and thus significantly impact the clinical treatment of challenging, patient-specific bone defects.