ABSTRACT As indicated by the 2019 Global Burden of Disease Study, although women, on average, have a longer lifespan than men, women typically experience worsened health outcomes as they age. Notably, postmenopausal women have a higher incidence of knee osteoarthritis (KOA) and present with more severe disease progression over time. However, it is still unclear whether these differences are genetic in nature, hormonal in nature, or a combination of the two. Here, we propose to investigate whether and how changes in the chondrocyte microenvironment—including soluble factors (Aim1) and extracellular matrix (ECM) stiffening (Aim2)—drive KOA pathogenesis in a sex-, gender- and age-dependent manner. Specifically, in Aim 1, we will mechanistically interrogate the direct role of soluble microenvironmental factors on the loss of cartilage integrity with aging and according to sex. In Aim 2, we will interrogate the contribution of ECM biophysical properties on the loss of cartilage integrity with aging and according to sex. We will accomplish these aims using technically innovative approaches, including: (1) an in vitro and in vivo heterosex biofluid exchange model, which will allow us to disentangle the multi-dimensional contribution of environmental factors on chondrocyte health and cartilage integrity over time; (2) an advanced network medicine approach integrated with machine learning algorithms to elucidate the sex chromosome- and sex hormone-dependent regulatory landscape; and (3) a physiologically relevant model of female aging by which menopause is chemically-induced in middle-aged mice. These conceptually and technically innovative studies will be carried out by an interdisciplinary team comprising a stem cell biologist (PI), a computational biologist (co-PI), and a bioengineer (co-I), the breadth of which enhances feasibility, impact, and likelihood for success. Successful completion of the aims as proposed will provide mechanistic insights into how sex and gender profiles impact cartilage physiology over time. Ultimately, we anticipate that the innovative research studies proposed will aid in the development of effective interventions that consider sex- and gender-specific variables to the benefit of our aging population.