Project Summary/Abstract Injuries to soft tissues represent 45% of all musculoskeletal injuries per year. Fatigue loading causes damage at the microscale to collagen fibrils, which makes the tendon more susceptible to rupture. Changes to the native tendon composition are often associated with injury risk. In the aging tendon, interfibrillar structures between fibrils (e.g., glycosaminoglycans (GAGs)) have been shown to decrease, in association with alterations to interfibrillar mechanics and rupture rates. While GAGs may not play a direct role in elastic mechanics, they have been postulated to promote fibril sliding by retaining water and increasing fibril spacing and lubrication, meaning that they provide an important load-bearing mechanism in tissues with aligned fibrils. This sliding mechanism may protect against repetitive, viscoelastic processes that cause tendon damage, namely fatigue, by reducing overall load to individual collagen fibrils. Yet, the connection between GAGs and fatigue-induced rupture remains unelucidated. The goal of this proposal is to define the multiscale interplay between GAGs, interfibrillar load transmission, and fatigue rupture in intact and healing mature and aged tendons. Our overall hypothesis is that GAGs modulate fibril spacing which enables sliding between aligned fibrils and protects against fatigue-induced microdamage and eventual rupture in intact and late-stage healing tendons. The proposed aims would innovate preclinical evaluations of multiscale tendon mechanics and augment the scientific understanding of micromechanical changes that precede injury in aging and healing tendons. The Aims are: Specific Aim 1: In mature and aged Achilles tendons, define the role of GAGs in preventing damage accumulation and eventual rupture during fatigue loading. Specific Aim 2: In the healing Achilles tendon, define the role of GAGs in preventing damage accumulation and eventual rupture during fatigue loading. In both aims, we will couple state-of-the-art biomechanical testing of a high-throughput rodent model with rigorous micromechanical and histological measures of interfibrillar sliding and structures. We will use findings from these techniques to inform and refine computational shear-lag models of tendons to further explore the role of GAGs in fatigue loading. These exciting and innovative studies will elucidate the role of GAGs in interfibrillar sliding, microdamage, and eventual rupture in Achilles tendons undergoing fatigue loading. Further, these studies will increase our understanding of mechanisms that lead to injury in aging and healing tendons, which is essential to our understanding of how tendon injuries occur, to improve interventions preceding injury, and enhance future therapeutic strategies following tendon injury.