SUMMARY Detection of Collagen Mechanical Damage using Collagen Hybridizing Peptides. Mechanical injury to load-bearing tissues leads to many clinically significant conditions (e.g. tendinosis, rotator cuff disease) but we have limited understanding of the injury process of tissues that are damaged by mechanical stress. The overall goal of the proposed research is to gain new understanding of the biomechanics of load bearing collagenous tissues by developing the collagen hybridizing peptide (CHP) technology into a new mechanical damage detection method. CHP has been reported to bind to denatured collagen strands originating from protease activity or by mechanical damage in a manner similar to primer binding to melted DNA during PCR. We propose to substantially expand the capabilities of CHP damage detection by developing new CHPs that are smaller for faster diffusion into dense musculoskeletal tissues and exhibit accelerated binding kinetics to allow faster damage reporting. We will also develop a new CHP that only fluoresces upon binding with collagen, eliminating the need to stain and wash tissues and enabling the CHP to serve as a damage gauge in overloaded tissues. We will then develop optimized protocols for the use of the existing and new CHPs, determine the relationship between collagen fibril strain and CHP binding in musculoskeletal soft tissues, and quantitatively compare CHP targeting to other techniques. Finally, we will apply CHP targeting to elucidate the relationship between tissue level mechanical loading and mechanical damage to collagen at the molecular level. We will focus on two important musculoskeletal tissues of considerable clinical relevance: tendons and articular cartilage. Considering the wide-spread impact of collagen damage in musculoskeletal injuries and diseases, in- depth understanding of the relationships between molecular level collagen damage and mechanical overloading will provide new insights into the biomechanics of load-bearing tissues as well as help develop new diagnostics and therapies for managing musculoskeletal disorders.