Project Summary Bone fragility fractures that occur in the absence of significant trauma are often associated with primary or secondary osteoporosis, and can result in serious patient morbidity and increased mortality rates. Prediction of bone fracture risk primarily relies on measures of bone mineral density (BMD), which is strongly correlated with bone strength, but not with fracture risk. Alternatively, Raman spectroscopy (RS), an optical technique that can provide information on mineral crystallinity, composition, and relative degree of mineralization (mineral/matrix ratio), as well as collagen composition and cross-linking, has emerged as a promising technique for assessment of bone strength and fracture risk. We have recently shown that RS can detect biochemical changes that occur in mouse models of rheumatoid arthritis (RA), glucocorticoid (GC)-induced osteoporosis (GIOP), and osteogenesis imperfecta, which correlated with independent measures of biomechanical strength and fracture toughness. We have also developed instrumentation to enable the first diagnostically-sensitive transcutaneous Raman measurements of murine bone on intact limbs, along with sophisticated algorithms to reduce optical contributions from overlying soft tissue, but these measurements were only made ex vivo on tissue specimens. In this application, we will develop new instrumentation and algorithms to adapt our transcutaneous RS measurements on live animals. More importantly, based on unpublished data demonstrating that bone ends (epiphyses) exhibit more discriminate RS differences than mid-shaft (diaphysis) regions, we will redesign the excitation/collection optics in our RS platform to provide a larger range of source- detector separation, greater variety in sampling depth, and ultimately improve the ability to resolve the spectral contributions from interfering soft tissues in the more anatomically and biochemically complex epiphyses regions (Aim 1). We will then validate and correlate regional (epiphysis versus diaphysis) transcutaneous Raman spectroscopy measurements with regional and whole bone mechanics (measures of bone quality) in juvenile, skeletally mature, and aged mice (Aim 2). Finally, the studies will demonstrate that our transcutaneous RS platform has the sensitivity to detect longitudinal reductions in bone quality in mouse models of RA and GIOP over time, and improvements in bone quality in response to anti-resorptive and anabolic treatments (Aim 3). Upon completion, the proposed studies will have validated a disruptive technology for pre-clinical, non-contact optical assessment of bone fragility and fracture risk, which are undetectable by standard metrics such as BMD. While successful completion of this work will yield a new instrument in our research toolbox to advance our understanding of mechanisms of osteoporosis and to evaluate efficacy of new drugs in preclinical models, we hope that the progress we make here will allow this non-invasive technology to be scaled-up and translated in the not too distant future as an experimental diagnostic tool in the clinic.