PROJECT SUMMARY/ABSTRACT The etiology of osteoarthritis (OA) is multi-factorial. Abnormal and excessive cumulative joint stress results in post-traumatic osteoarthritis (PTOA). Approximately 30% of knee OA in Veterans is attributable to occupational activities, particularly jobs requiring kneeling or squatting in combination with heavy lifting, such as sport activity injuries. Currently, there is no effective therapy for OA patients. Recently, the stem cell therapy has shown promise to regenerate the damaged joint tissue. In our previous study, adipose-derived stem cells (ASCs) show great promise as therapeutic agents in regenerative medicine because of their multi-lineage potential, immunosuppressive activities, limited immunogenicity, and relative ease of growth in culture. However, there are several concerns that impede the clinical use of stem cell therapy in the inflammatory joint environment such as apoptosis, dosing, timing of intervention, homing efficacy, and route of delivery of ASCs. We have also found that NF-κB inhibitors such as TPCA-1 decrease inflammation in mechanically injured knee joints using our established PTOA mouse model and in vitro model. In this project, we aim our studies based on two points: 1) Pre-treatment of exogenously derived ASCs with antioxidant (such as Vitamin-E) before injection into the joint can lead to cyto-protective effects and resistance to apoptosis and toxic inflammatory factors after transplantation. 2) TPCA1-nanosome can improve the harsh condition in the arthritic knee joint by anti-inflammatory mechanism before transplantation of an exogenous stem cell. Therefore, we will show the synergistic effect of this two-step therapeutic application (anti- inflammatory nanosome treatment of the arthritic joint followed by transplantation of the Preconditioned ASCs). We have two aims: (1) Investigate the cytoprotective effects and therapeutic potential of preconditioned ASCs in a model of PTOA. In this aim, we will investigate the therapeutic effect of the antioxidant (Vit-E-Ns) pre-treated ASCs in vivo using our PTOA mouse model of knee overloading. In order to tailor this approach to the appropriate veteran population (active vs retired) we will use aged mice to investigate whether they exhibit different healing responses and mechanisms relative to younger mice. We will confirm the persistence of the ASCs in the joint, correlating implantation levels with the reduction and repair of damaged cartilage as well as optimizing the cell number and treatment interval. We will examine localization of the ASCs in the joint and assess joint inflammation and cartilage integrity. We will investigate treatment-associated changes in the biomechanical properties of subchondral bone and cartilage and its effects on pain-related behavior through functional analyses. We will also investigate the recovery mechanism using an ASC-chondrocyte co-culture system. (2) Demonstrate the synergistic therapeutic efficacy of the two...