Project Summary Bone metastasis is a major cause of morbidity and mortality in cancer survivors. It is closely linked to advanced skeletal aging, which is driven by the accumulation of aged (senescent) cells that secrete proinflammatory molecules. Pharmacologically delaying bone aging remains the most effective preventive strategy against bone metastasis. Osteoporosis drugs effectively impede bone aging and metastasis, but their benefits are transient. Senolytic drugs are a new class of drugs that have shown compelling preclinical evidence in delaying skeletal aging. However, their clinical adoption has been slow due to the heterogeneous cellular senescence and tissue- specific efficacy. This proposal aims to develop and validate demineralized bone paper (DBP)-based human bone metabolic and senolytic assays that support bone-targeting drug discovery and treatment regimens. DBP is an osteoid-inspired thin slice of demineralized compact bone matrix that preserves the intrinsic collagen structure of bone while remaining accessible for microscopic imaging. DBP can be produced in large quantities and attach stably to tissue culture plastic. This feature effectively enables DBP to receive mechanical stress transmitted via vibration. Attaching DBP in a standard 96-well plate provides a unique opportunity to develop standardized, functional, and analytical human bone models and phenotypic assays. In Aim 1, we will develop humanized bone metabolic assays. We will humanize DBP-based bone models with human bone marrow-derived mesenchymal stem cells and human CD14+ monocytes, mimicking bone remodeling cycles. Next, we will create anabolic and catabolic assays using the models and validate with known agents. Finally, we will test whether the models can recapitulate osteoporosis treatment outcomes using Denosumab and Zoledronate. In Aim 2, we will develop humanized bone senolytic assays. We will first introduce pre-senescent osteoblasts and co-culture with human CD14+ monocytes to create an aged bone model. Next, we will validate that senescent models increase osteoclast differentiation and mineral resorption. We will then test a model senolytic drug therapy (Dasatinib and Quercetin) to mitigate negative impacts. Finally, we will investigate whether vibrational mechanoculture affects senolytic drug responses. The proposed DBP-based models will provide predictive bone assays for drug screening, which could significantly impact the field by accelerating the identification of new anti-bone-aging drugs and improved treatment strategies. Overall, this proposal is a significant contribution to the field of bone research and has the potential to make a major impact on the lives of cancer survivors.