PROJECT SUMMARY/ABSTRACT Approximately 30% of the 35 million Americans with temporomandibular joint (TMJ) disorders experience TMJ disc degeneration and mechanical dysfunction, and around 10% of these cases require surgery. Replacement of the fibrocartilage using muscle/fascia flaps or fat and/or dermis grafts is common, but also comes with complications necessitating the search to find alternatives. The research to replace the TMJ disc involves the need to replicate its morphological, biological, and mechanical properties. Utilizing a TMJ disc allograft would be an ideal option given its natural properties closely resembling those of the original tissue. The use of fresh allograft TMJ discs in animals has been studied and a similar synovial joint, the knee meniscus, already utilizes allograft transplantations. Limited refrigeration storage periods of fresh TMJ discs and tissue damage during conventional cryopreservation by slow-freezing are two of the more significant reasons that TMJ allografts are not a treatment option. The use of slow-freezing leads to the formation of ice crystals inside the cells and extracellular matrix causing damage and cell death. To overcome these limitations this project proposes the use of vitrification, which avoids ice crystal formation by transforming the liquid solution into a glassy solid using high concentrations of cryoprotectants (CPAs), with a commonly used solution being the 55% multi-CPA cocktail known as VS55. Given that high concentrations of CPAs are required for vitrification procedures to avoid ice crystal formation, an essential challenge lies in finding methods to minimize the cytotoxicity of CPAs while ensuring their sufficient diffusion into the tissue. Specifically, we propose the following aims: Aim 1: To investigate different VS55 loading periods on cell viability, ECM integrity, and mechanical functionality in vitrified TMJ discs and Aim 2: To investigate different DP6 (a lower toxic alternative compared to VS55) loading periods on cell viability, ECM integrity, and mechanical functionality in vitrified TMJ discs. The proposed work will enable us to establish an optimal vitrification procedure as an effective cryopreservation technique for the long-term storage of donor TMJ discs. Through our exploration of vitrification, we expect to significantly improve the prospects of successful donor TMJ disc transplantation in the future. This fellowship will assist me in advancing my understanding of cryopreservation and therapeutic approaches designed to treat the loss of TMJ discs, as well as provide me with cutting-edge training that will support my development as a clinician scientist.