|The temporomandibular joint (TMJ), associated with everyday activities, such as chewing, yawning, talking, and laughing, is one of the most complex but least studied joints in the musculoskeletal system. Patients suffering from TMJ disorders (TMDs) may experience a variety of agonizing symptoms, such as earaches, headaches, neck pain, and difficulty opening the mouth, while current treatments are inefficacious owing to a poor understanding of TMJ disorder pathologies. Prior to this thesis, human umbilical cord mesenchymal stromal cells (hUCMSCs), identified as multipotent cells only in this decade, had not yet been used for TMJ tissue engineering. Hence, this thesis proposed a revolutionary tissue engineering approach in which hUCMSCs and mature hyaline cartilage cells, scaffolds, and growth factors were integrated to create TMJ condylar bone and cartilage in vitro to substitute for deteriorated native tissues. hUCMSCs were successfully differentiated along chondrogenic and osteogenic lineages in a 3D biomaterial-based environment, supporting the feasibility of using these cells for TMJ cartilage and bone tissue engineering. In TMJ cartilage regeneration, hUCMSCs demonstrated significant advantages over both mature TMJ cells and human bone marrow mesenchymal stromal cells (hBMSCs), with faster cell proliferation and superior biosynthesis. As an additional alternative, hyaline cartilage cells also surpassed TMJ cells in that they produced considerably more extracellular matrix. Following the initial efforts, the cell culture environment was refined, including cell seeding densities and signaling strategies. Higher cell seeding densities (25 million cells/ml) were recommended for both cartilage and bone tissue engineering, mainly due to their benefits to differentiation and biosynthesis. Insulin-like growth factor I (IGF-I) enhanced the chondrogenesis of pre-differentiated hUCMSCs while having no effect on osteogenically induced hUCMSCs. Therefore, the work on the successful differentiation of hUCMSCs in 3D biomaterials has been pioneering, and the culture parameters for in vitro TMJ tissue engineering have been refined. Moreover, this innovative work has tremendous implications for a broader area (e.g., musculoskeletal tissue engineering).