Project summary Temporomandibular joint disorders (TMJDs) are estimated to affect over 10 million Americans as per NIDCR. Total 80 - 90% of symptomatic TMJDs patients have internal derangement (ID), also referred to as disc displacement, which is highly associated with osteoarthritis (OA) that may necessitate surgical treatment. Previous attempts to replace the TMJ disc with alloplastic and/or synthetic grafts have failed, resulting in further joint degradation. Thus, regeneration of TMJ disc has recently emerged as an alternative approach to overcome limitations of current treatments for TMJ disorders. In our preceding studies, we developed anatomically correct 3D-printed polycaprolactone (PCL) scaffolds with native-like anisotropic microfiber orientation. To engineer the native-like heterogeneous fibrocartilage, connective tissue growth factor (CTGF; profibrogenic cue) and transforming growth factor beta 3 (TGF?3; chondrogenic cue) were spatially embedded in the scaffolds as encapsulated in poly(lactic-co-glycolic acids) (PLGA) microspheres (?S). The spatiotemporal release of CTGF and TGF?3 guided recruitment of TMJ syMSCs, followed by spatially controlled fibrocartilaginous differentiation toward regeneration of TMJ in rabbits and mini-pigs. Despite the promising in vivo outcome, our CTGF/TGF?3 ?S-embedded scaffolds encountered few outstanding translational challenges for TMJ discs regeneration, including PLGA degradation-derived acidic environment, a notable interspecies variance in the in vivo degradation rate of scaffolds, and potential side effect of over-physiological dose of growth factor. To address these issues, here we propose to develop and validate a novel combination of pharmaceutical small molecules to replace CTGF and TGF?3 as incorporated in bioactive scaffolds, to refine the in vivo degradation rate as balanced with de novo tissue formation through our advanced imaging modality, and then to promote regeneration of TMJ discs in a pre-clinical large animal model. Our preliminary study identified novel small molecules that are safe and highly efficient and specific for promoting fibrocartilaginous differentiation of TMJ- derived syMSCs. We also achieved a precisely controlled delivery of the small molecules in 3D-printed TMJ disc scaffolds by adopting a self-assembling multi-domain peptide (MDP) hydrogel as a delivery vehicle. We also devised a highly efficient and reliable imaging modality that will enable to track in vivo scaffold degradation and new tissue formation. We will perform a comprehensive comparative study between small molecules and CTGF/TGF?3 as control-delivered in our scaffolds regarding local/tissue pH change, cytotoxicity, degradation and tissue formation in our TMJ disc engineering model in vitro. We will conduct a comprehensive in vivo study to balance scaffold degradation with tissue regeneration. The degradation rate will be controlled by applying surface micro-porosity, and in vivo tracking of scaffold degradation as well as fibrocartilage regeneration will be achieved via our minimally invasive imaging modality.