The role of MSCs in immunoregulation of macrophages during osteogenesis

Osteoimmunology as an interdisciplinary research principle governing the cross-talk between the skeletal and immune systems. Better understanding of how these two systems operate is likely to lay the groundwork for biological therapies of bone related diseases. Mesenchymal stromal cells (MSCs) are self-renewing and pluripotent cells capable to differentiate into osteoblasts. Previous studies have shown that undifferentiated MSCs possess remarkable immunoregulatory properties through inhibiting the proliferation and activation of the major immune cell populations. However, it is unknown whether MSCs have the same immunoregulatory properties during osteogenic differentiation. We have reported for the first time that undifferentiated MSCs and osteogenically differentiated MSCs (OMSCs) have different effect on cell recruitment. Macrophages as the precursors of osteoclasts and immune cells were used as the effector cells for cell migration. We found that cell motility was enhanced by the OMSC conditioned medium (OMSC-CM) compared to the MSC conditioned medium (MSC-CM). It was noted that OMSCs also regulated the local immune responses by modulating cell morphology and elicited significant effects on macrophage activation, resulting in the up-regulation of pro-inflammatory cytokine expression. These results indicate that OMSCs are actively involved in bone formation by regulation of cell migration and activation. In the second part of our study, the potential factors secreted by OMSCs contributing to cell migration were evaluated through cytokine array analysis. Our results have demonstrated that OMSCs could regulate macrophage motility through the secretion of VEGF, which is involved in bone formation due to the fact that impaired bone formation was observed with the neutralization of VEGF in skull defects. In addition, VEGF secreted by OMSCs slightly activated the macrophages by up-regulation of pro-inflammatory cytokine expression. This study explores the key linkage between the skeletal and immune systems and expands the scope of osteoimmunology. The last part of our study has investigated the contribution of secreted factors from OMSCs to immune cell activation through the study of relationship between the vascular endothelial growth factor (VEGF) and the receptor activator of nuclear factor kappa-Β ligand (RANKL). Our data suggest that the inhibition of VEGF impaired osteogenesis and RANKL expression. In addition, RANKL was closely correlated to iNOS positive cells in vivo. In vitro study found that RANKL could trigger iNOS expression in macrophages which were involved in osteogenic differentiation of MSCs. RANKL induced macrophages showed different expression pattern in terms of cytokine expression compared with LPS and IFN-γ. This study emphasizes the involvement of VEGF and RANKL in macrophage polarization under physiological conditions in comparison to LPS and IFN-γ, which are derived from pathogens. Taken together, our study reveals a quite variable and multifaceted profile of osteogenically differentiated MSCs which enhance bone formation through induction of macrophage migration and activation. These findings enrich our understanding of osteoimmunology and bone remodelling, which will pave the theoretical principles for cell- based therapy in bone defects