Therapeutic Potential of Mesenchymal Stem Cell‐Conditioned Medium via Macrophage Subset Regulation in a Mouse Model of Arthritis‐Associated Pain

Osteoarthritis (OA) is a leading cause of chronic pain and disabilities. Current treatments offer limited efficacy and are often associated with adverse effects. Therefore, novel therapeutic strategies are urgently needed. Mesenchymal stem cell-conditioned medium (MSC-CM) possesses immunomodulatory properties and has been shown to suppress macrophage (MΦ) activity, which plays a key role in arthritis pain. However, the effect of MSC-CM-mediated MΦ regulation on arthritis-induced pain remains unclear. In this study, we investigated the analgesic effects of MSC-CM in a mouse model of adjuvant-induced arthritis (AIA), established by the injection of complete Freund's adjuvant (CFA), and examined the involvement of MΦs in mediating these effects. MSC-CM was administered as a single intravenous injection via the tail vein 1 d after the CFA injection. Mechanical and thermal hyperalgesia were evaluated using von Frey filament and the Hargreaves test, respectively. MSC-CM significantly alleviated both mechanical and thermal hyperalgesia but had no effect during the recovery phase. On Day 3 post-CFA injection, when the analgesic effect was most pronounced, flow cytometric analysis revealed that MSC-CM selectively reduced pro-inflammatory M1 MΦs at the site of inflammation without affecting pan-MΦs or anti-inflammatory M2 MΦ populations. Additionally, depletion of MΦs using clodronate liposomes attenuated hyperalgesia in AIA mice. These findings indicate that hyperalgesia in AIA mice was MΦ-dependent and that MSC-CM exerted its antihyperalgesic effects by suppressing MΦ activation at inflammatory sites, highlighting its potential as a novel therapeutic approach for inflammatory pain.