Project Summary Tendon overuse initiates mechanical damage that leads to chronic tendinosis (degeneration) and tendinopathy (clinical presentation with pain), which are common and notoriously difficult to treat. Despite the widely accepted role of loading in tendinosis, the structural, mechanical, and cellular mechanisms by which loading leads to initiation and progressive damage in tendinosis remain unknown. We hypothesize that overload causes micro-scale structural and mechanical damage, which alters load transmission to cells, driving the multi-scale structural and molecular progression of tendinosis in a vicious cycle. To determine the mechanisms involved in tendinosis, a preclinical in vivo animal model of tendon overuse and multiscale assessments of tendon structural and mechanical damage and interrogation of cellular mechanotransduction and intracellular signaling mechanisms are all required. This is because the physiological processes in tendinosis involve tissue-scale tendon loading that is transferred to the micro-, nano-, and molecular-scale, where microstructural damage and cellular mechanotransduction signaling occurs. Our team has recently established a model of tendinosis using rat synergist ablation (SynAb), where we remove the Achilles tendon, which overloads the synergistic plantaris tendon without directly injuring it. Our pilot data exhibit hallmark features of human tendinosis (increased area, collagen disorganization, proteoglycan accumulation, collagen Type III production, and reduced tensile modulus). Our long-term goal is to enable interventions for tendon regeneration and rehabilitation to treat tendinopathy and prevent its progression. The objective of this proposal is to determine the mechanisms responsible for onset and progression of tendinosis in the SynAb model of tendon overuse in the following aims: Aim 1: Assess the multiscale structural changes following the onset and progression of tendinosis. Aim 2: Quantify the multiscale mechanical properties and damage following the onset and progression of tendinosis. Aim 3: Interrogate changes in tenocyte mechanoresponse and cytoskeleton during tendon overload. This study will determine the mechanisms responsible for the multiscale damage in overuse tendinosis and establish these in the context of key hallmarks of human tendinosis using a preclinical in vivo model. Quantifying multiscale damage and cellular mechanisms by which loading leads to tendinosis is critical for designing and evaluating interventions to prevent and treat tendinopathy.