SUMMARY Rotator cuff tendon tears account for more than 4.5 million physician visits and over 300,000 surgical repairs annually in the US. The healing rates of the tendon-bone interface, i.e., enthesis, remain low, despite continual improvements in fixation techniques. Moreover, pre-existing degeneration at the rotator cuff enthesis puts an estimated 17 million Americans at increased risk for rotator cuff impairment and tearing. A growing body of evidence indicates that cigarette smoking is an important risk factor. CDC data show that almost 13% of US adults smoke cigarettes, yet South Carolina rates are even higher at nearly 18%, with high school youth smoking cigarettes in SC at more than 3 times the US rate. Despite this significant impact, we still lack a fundamental understanding of the effects of cigarette smoking on the mechanical properties of the enthesis and the biomechanic mechanisms. Obstacles in formulating such understanding are partly attributed to (i) challenges in creating a realistic smoke exposure environment using well-controlled animal studies and (ii) the difficulties in characterizing the small-scale structural changes and correlating the structural changes with the deterioration of mechanical properties. We propose to utilize our custom-built automatic smoking chamber that resembles realistic cigarette smoking on a rat model, plus adopt an integrated experimental and computational approach to systematically understand the structure-function relationship of rat supraspinatus tendon-humerus enthesis and how it is impacted by cigarette smoking. The proposed studies will test the hypothesis that smoking directly affects the structure of collagen fibers plus the mineral gradient and distributions at the enthesis, all of which we further hypothesize will lead to deterioration of the enthesis by changing the microscopic deformation patterns. The specific aims are (1) to characterize the impact of smoking on the mechanical properties of the rotator cuff tendon-bone enthesis; (2) to delineate the changes from smoking on the multiphasic structures at the tendon- bone enthesis; (3) to construct a multiscale modeling framework that bridges the structures with the mechanical properties. We will integrate tissue biomechanics, bioimaging, and multiscale modeling to characterize the structure-function relationship of the enthesis, provide novel mechanistic data that will inform rotator cuff injury risk associated with cigarette smoking, and understand the underlying mechanisms. Once this approach is successfully applied, the tools and insights from this project can be transferred to other soft tissue-bone interfaces. Moreover, the new knowledge generated from this project, particularly the degenerative impact of smoking on rotator cuff tendon-bone enthesis, will lay the foundation for innovating treatment strategies for repairing the interfaces and improving the surgical outcomes of high-risk and/or rural area patients.