Abstract The temporomandibular joint (TMJ) controls the movement of the jaw but is susceptible to acquired diseases, including osteoarthritis (OA). Osteophytes, referred to as fibrocartilage-capped bony spurs, are one of the radiographic hallmarks of TMJ degenerative disease and cause skeletal deformities, chronic pain, and limited condyle movement. Current treatments are limited, and patients may undergo reparative surgical procedures, which are not always remedial. The etiology and pathogenesis of osteophyte formation in TMJ OA remain unresolved and interfere with basic, translational, and clinical progress toward new effective treatments. Recently, arthropathy, including in the TMJ, was reported for the first time in a patient with compound heterozygous mutations in the PRG4 gene. Our preliminary studies show that Prg4-mutant mice reproduce some key phenotypes in TMJ OA patients, including synovial hyperplasia and osteophytes. Whereas heterozygous Prg4-deficient (Prg4+/-) mice did not exhibit a strong TMJ phenotype, osteophytes did form in homozygous Prg4-null (Prg4-/-) mice, initiating ectopic chondrogenesis. The latter data indicate that local normal anti-chondrogenic mechanisms had been deranged, possibly causing greater availability of chondrogenic factors and leading to osteophyte onset. We tested this novel thesis and found that before osteophyte formation, the expression of the heparan sulfate (HS) synthesizing gene EXT1 was downregulated in the TMJ fibrocartilage. In addition, we found that the local levels of heparanase (HSPE), the primary enzyme responsible for extracellular HS degradation, were markedly increased in osteophyte-forming regions of TMJs. These preliminary data suggest that local decreases in HS and increases in HSPE cause a significant drop in HS levels and create a robust prochondrogenic-inducing environment. In support of this thesis, BMP signaling was more greatly distributed within the mutant condyles and developing osteophytes. These and other data lead to our central hypothesis that a steep local loss of HS triggers osteophyte formation. Aim 1 will test whether conditional Ext1 ablation causes osteophyte formation or worsens the TMJ phenotype in Prg4+/- mice. We will conduct single-cell RNAseq studies to assess cellular phenotypic characteristics, developmental trajectories, signaling pathways, and networks during osteophyte formation. Aim 2 will test whether Bmpr1a ablation will inhibit osteophyte formation in Prg4-null and other mutant mice above. To expand treatment options and make them clinically relevant, we will test whether administering a BMP signaling antagonist LDN-193189 prevents or inhibits osteophyte development. This project is based on new data and insights into the mechanisms of osteophyte formation in the TMJ and will test the efficacy of drug therapy. Given its novelty, the project is in its early stages but will undoubtedly have major and broadly relevant implications for basic research and t...