TR&D 3: Bioprinting for Complex Scaffold Fabrication Project Summary Most biological tissues are composed of a complex microarchitecture with well-defined extracellular matrix (ECM) composition, mechanical properties, and cell populations. Due to this high level of complexity, we have employed a multi-material 3D printing system with the potential to recapitulate heterogeneous tissue by printing material composites and depositing growth factors in a gradient organization to address three Specific Aims: (1) printing polymer/ceramic composites (of poly(ε-caprolactone) (PCL), poly(propylene fumarate) (PPF), hydroxyapatite (HA), and β-tricalcium phosphate (β-TCP)), (2) controlling the deposition and release of bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) from PPF-based scaffolds for vascularized bone repair in a femoral defect, and (3) depositing insulin-like growth factor-1 (IGF-1) and transforming growth factor β-1 (TGF-β1) in a gradient organization within the 3D printed (3DP) PCL-based scaffolds for osteochondral tissue repair. The rationale for the use of a multi-material 3D printing system is the ability to print a wide variety of materials (polymers, ceramics, and bioactive molecules), the accessibility and flexibility of deposition systems that can be incorporated with the printer, and the ability to establish the parametric space for printing various polymers within the realm of open-source technologies. Due to the flexibility of this system to accommodate a variety of tissue types and constructions, a wide variety of collaborative and service projects are proposed to be beneficial for TR&D3. The versatility of the proposed multi-material printing system provides a platform to modify scaffold design criteria for different materials, bioactive molecules, or cell types. We offer expertise in the processing of polymers (PCL, PPF) and ceramic pastes (HA, β-TCP) for extrusion-based printing and will work with the other TR&Ds to optimize accuracy of printing these and other biomaterials for use in dynamic culture systems (TR&D1) and controlled cell printing (TR&D2). To extend the relevance of our multi-material printing technology, we will utilize alternative materials developed by the Collaborative Projects, such as injectable, guest-host hydrogels and native tissue-derived ECM components for the 3D printing of osteochondral scaffolds, as well as injectable, thermoresponsive polymers and alginates for the 3D printing of bone scaffolds. Through these interactions, we will offer our expertise in fabricating gradient heterogeneities within these constructs to direct cellular and tissue responses. We will also p...