A single joint injury can result in the development of post-traumatic osteoarthritis (PTOA). Central to loss of joint function is the progressive degeneration of articular cartilage. While surgical procedures can correct structural defects, there are no treatments that can protect chondrocyte metabolism and viability in the weeks after an injury. Preclinical studies have shown that mesenchymal stem/stromal cells (MSCs) grown in vitro can have chondroprotective effects in animal models of PTOA; these effects are now thought to be mediated by MSC paracrine signaling following intra-articular delivery. Recently, MSC-derived extracellular vesicles (MSC-EVs) have been shown to stimulate pro-regenerative effects equivalent to their donor cells in several non-skeletal injury models, suggesting they are an important component of MSC paracrine actions. The potential of MSC-EVs for preventing or delaying PTOA may depend on their ability to deliver signals directly to chondrocytes, which requires transport through the dense, negatively-charged matrix of cartilage. While EV size and net charge suggest limited penetration within healthy cartilage, their precise surface composition may influence cartilage uptake and diffusion. Moreover, cartilage matrix changes that occur during the acute inflammatory period after a joint injury can alter the transport of larger solutes, and EVs may respond similarly. If MSC-EVs can deliver signals that improve chondrocyte function before irreversible changes occur to the cartilage matrix, they can be used to restore tissue homeostasis during the critical period following joint trauma. This project will better define the therapeutic potential of MSC-EVs for PTOA through an improved understanding of the factors regulating their cargo delivery to chondrocytes. We will first test whether cartilage glycosaminoglycan depletion, a more reversible change to the cartilage matrix than collagen loss, impacts MSC-EV cargo delivery (Aim 1). We will also determine how delivery is influenced by MSC-EV surface charge and altered electrostatic interactions with the cartilage matrix (Aim 2). Finally, we will identify chondrocyte genes that control MSC-EV cargo delivery through a genome-wide gain-of-function screen (Aim 3). In the short term, this project will lay the groundwork for studying potential EV modes of action for the prevention of PTOA; in the long term, it will inform the design of synthetic vesicles for delivering therapeutic molecules to synovial joint cells.