CD40 stimulation improves osteogenesis from mesenchymal stem cells via the activation of TGF-β

The immune-skeletal interface is increasingly seen as an important regulator of bone health. CD40, a well-known costimulatory receptor in adaptive immunity, has emerged as a significant player in bone biology. While CD40 signaling affects blood cell formation and bone remodeling, its role in mesenchymal stem cell (MSC) decisions remains unclear. In this study, we show that murine bone marrow-derived MSCs express functional CD40 at both the transcriptional and translational levels. When CD40 was engaged with an agonistic antibody, it greatly improved osteogenic differentiation, shown by increased alkaline phosphatase (ALP) activity, faster calcium mineralization, and early expression of osteogenic markers like osteopontin. Further analysis showed that CD40 stimulation boosted osteogenesis by activating the TGF-β/Smad2/3 pathway, along an increase in TGF-β receptor 1 expression, while MAPK signaling remained unchanged. Additionally, engaging Toll-like receptor 9 (TLR9) with CpG oligodeoxynucleotides raised CD40 expression in MSCs. The concerted actions of CpG and CD40 stimulation further enhanced osteogenesis, shown by higher ALP activity, mineral deposition, and Runx2 expression. Interestingly, the anti-CD40 driven enhancement in osteogenesis both with and without CpG was not restricted to murine MSCs alone, rather, the human gingival-derived MSCs (GMSCs) also showed the same, suggestive of species-independent efficacy. These findings delineate the new found role of CD40, an essential immunoregulatory receptor that may establish a connection between immune activation and bone formation. Our research highlighted the plausible potential of targeting CD40 and innate immune pathways to improve MSC-driven bone regeneration in conditions like osteoporosis, fracture repair, and bone loss related to immune dysfunction.