Project Summary/Abstract More than 300,000 rotator cuff surgeries occur annually in the US. However, treatment is often complicated by disorganized collagen matrix formed via fibrosis and results in high re-tear rates. Tendon tissue engineering seeks to solve the problem using biomaterials to promote neo-tendon formation to augment repair or regenerate tendon. However, while current biomaterials provide the opportunity to improve tendon healing, they frequently still exhibit fibrosis in preclinical studies. Therefore, a critical need exists to understand the mechanisms of aligned collagen formation when designing biomaterials for tendon tissue engineering. Matrix architecture and transient receptor potential cation channel subfamily V member 4 (TRPV4) regulate aligned collagen formation during tenogenesis in vitro, but the mechanism remains to be determined. Recently, TRPV4 stimulation was found to induce nuclear localization and activation of transcriptional co-activators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) in keratinocytes and myofibroblasts during fibrosis. YAP expression is upregulated during tendon development, a process characterized by aligned collagen formation, and in response to physiological mechanical stimulation, suggesting it could play an important role in tendon. Both TRPV4 and YAP/TAZ are dysregulated in lung, liver, kidney, and cardiac fibrosis. However, the role of TRPV4 and YAP/TAZ, and their interactions, have not been investigated in tendon fibrosis or during engineered tendon development. The overall hypothesis is that TRPV4 regulates collagen alignment via YAP/TAZ during tenogenesis on biomaterials and stimulates neo-tendon formation to a similar extent as tensile loading. In contrast, dysregulation of this relationship between YAP/TAZ and TRPV4 will promote fibrotic matrix deposition. Aim 1 will compare the role of YAP/TAZ and TRPV4 in healthy and fibrotic rotator cuff tendon. Aim 2 will investigate the interaction between TRPV4 and YAP/TAZ in aligned collagen formation during tenogenesis on electrospun biomaterials in ‘healthy’ and ‘fibrotic’ conditions. Aim 3 will evaluate how TRPV4 stimulation affects neo-tendon formation on biomaterials. This information will directly inform planned preclinical studies of rotator cuff repair initially in rodent models, before, if successful, moving to large animal models and clinical translation. Completing the proposed studies will provide me with the opportunities and training to support my career goals and develop the skills I need to achieve my long-term goal of an independent faculty career at a research-intensive institution.