PROJECT SUMMARY Temporomandibular joint (TMJ) disc degeneration and perforation are the leading causes of debilitating TMJ disorders in people, while current treatments remain palliative at best. Novel tissue-engineering methods using allogeneic chondrocytes can produce tissue akin to native in terms of biological and mechanical properties. Little attention, however, has been given to the immunological aspects of the engineered tissue-recipient interface. Understanding and defining key parameters that affect this interface are critical in the rapidly developing field of tissue-engineering and TMJ disc regeneration. MHC class I compatibility is one of the major parameters defining acceptance of the tissue implant. Chondrocytes constitutively express MHC I and thus can be identified as non-self and subsequently destroyed by CD8 cytotoxic T cells. The latter premise challenges the conventional concept of immunoprivileged status of cartilage tissue. Because MHC compatibility in tissue transplantation has not been explored in the context of TMJ or its disc, our first aim will determine the relevance of MHC class I matching in TMJ disc repair using allogenic cell-sourced tissue-engineered cartilage. We postulate that tissue-engineered constructs sourced from MHC I-matched chondrocytes will have better long-term outcomes in the restoration of TMJ disc defects as compared to constructs sourced from mismatched chondrocytes. Further, by dissecting the type of host inflammatory response towards TE cartilage constructs in MHC I matched versus mismatched minipig recipients, we will determine if the type of immune response can predict long-term outcomes of implantation in the TMJ disc. MHC I silencing or knockout has not been attempted in primary chondrocytes but was achieved successfully in other types of primary cells. Our specific aim two will generate MHC I-null primary chondrocytes by knocking out the beta 2 macroglobulin gene using CRISPR/Cas9. We hypothesize that allogeneic lymphocyte proliferation will be decreased in coculture with MHC I-null chondrocytes as opposed to WT chondrocytes and that mechanically robust and biologically functional implants can be manufactured from MHC I-null chondrocytes. If successful, this strategy will provide a novel platform for the manufacturing of immunouniversal TMJ cartilage implants. We propose to carry out the proposed experiments in the minipig animal model, which is a well- established large animal model for translational studies of human TMJ. Our team has ample experience and expertise in working with this model. In summary, this project will deliver essential information on the immunology of the TMJ compartment and will boost the clinical translation of regenerative modalities of the TMJ disc.