Project Summary/Abstract Tendon and ligament injuries are a common cause of disability and pain. In many cases, the injured tissue cannot be repaired directly and must be replaced with a graft material. Ideally, a tissue engineered biomaterial could be used for this purpose; however, no tissue engineered construct has been successfully used to reconstruct human tendon or ligament ruptures. A primary reason for the failure in producing successful tendon replacements is that most tissue engineering approaches do not replicate normal tendon development. Scaffold-free techniques based on cellular self-assembly are able to generate tissues that that match the structure and mechanics of early embryonic tendon. However, they are unable to undergo a critical phase in late tendon development where the collagen fibrils elongate and fuse together generating a substantially stiffer and stronger material. One reason for this is that the constructs lacked the mechanical stimulation normally provided in vivo by muscles. In fact, chick embryo muscle activity peaks during late tendon development and this muscle activity is responsible for increasing the modulus of embryonic tendons. Nevertheless, while mechanical loading of constructs does improve tissue mechanics, they still fail to match the order-of-magnitude increase in mechanical properties observed in embryonic chick tendons. A critical barrier is the lack of knowledge regarding the mechanotransduction mechanisms that determine the cellular response to mechanical stimulation and drive late tendon development. Understanding how tendon cells respond to mechanical stimuli due to not only muscle loading but also the local changes in tissue structure and mechanics that occur during development is necessary to develop biomaterials that can successfully replicate tendon function. Therefore, the objective of this project is to identify the mechanotransduction mechanisms that mediate the multiscale changes in tissue structure and mechanics observed during late tendon development. Specifically, this project will (1) identify the multiscale structural and mechanical changes that occur during late tendon development and (2) determine the mechanotransduction mechanisms driving these changes. The overall hypothesis is that cells sense mechanical stimuli through a combination of cell-cell and cell-matrix interactions and that these mechanotransduction events are essential for driving proper tendon development. This will be evaluated by inhibiting embryonic muscle activity and perturbing mechanotransduction signaling in embryonic tendons via site-specific gene knockout during ex ovo culture of chicken embryos. The effects of these manipulations on tendon structure and mechanics will be determined by a novel combination of multiscale mechanical testing, computational modeling, and ultrastructural imaging. This work is the first investigation of the mechanotransduction mechanisms driving the structural and mechanical changes observed during late tendon development. The findings will provide the foundation for enhancing tissue engineered constructs to develop biomaterials that can successfully replace diseased tendons and ligaments.