T cells and macrophages jointly modulate osteogenesis of mesenchymal stromal cells

Abstract Approximately 5%–10% of fractures go on to delayed healing and nonunion, posing significant clinical, economic, and social challenges. Current treatment methods involving open bone harvesting and grafting are associated with considerable pain and potential morbidity at the donor site. Hence, there is growing interest in minimally invasive approaches such as bone marrow aspirate concentrate (BMAC), which contains mesenchymal stromal cells (MSCs), macrophages (Mφ), and T cells. However, the use of cultured or activated cells for treatment is not yet FDA‐approved in the United States, necessitating further exploration of optimal cell types and proportions for effective bone formation. As our understanding of osteoimmunology advances, it has become apparent that factors from anti‐inflammatory Mφ (M2) promote bone formation by MSCs. Additionally, M2 Mφ promote T helper 2 (Th2) cells and Treg cells, both of which enhance bone formation. In this study, we investigated the interactions among MSCs, Mφ, and T cells in bone formation and explored the potential of subsets of BMAC. Coculture experiments were conducted using primary MSCs, Mφ, and CD4+ T cells at specific ratios. Our results indicate that nonactivated T cells had no direct influence on osteogenesis by MSCs, while coculturing MSCs with Mφ and T cells at a ratio of 1:5:10 positively impacted bone formation. Furthermore, higher numbers of T cells led to increased M2 polarization and a higher proportion of Th2 cells in the early stages of coculture. These findings suggest the potential for enhancing bone formation by adjusting immune and mesenchymal cell ratios in BMAC. By understanding the interactions and effects of immune cells on bone formation, we can develop more effective strategies and protocols for treating bone defects and nonunions. Further studies are needed to investigate these interactions in vivo and explore additional factors influencing MSC‐based therapies.