Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells

Background: The mechanisms by which macrophage phenotype contributes to mesenchymal stem cells (MSC)-mediated bone repair remain unclear. In this work, we investigated the influence of factors released by human macrophages polarized to a pro-inflammatory or an anti-inflammatory phenotype on the ability of human MSC to attach, migrate, and differentiate toward the osteoblastic lineage. We focused on the role of TNF-α and IL-10, key pro-inflammatory and anti-inflammatory cytokines, respectively, in regulating MSC functions.

Methods: MSC were treated with media conditioned by pro-inflammatory or anti-inflammatory macrophages to study their influence in cell attachment, migration, and osteogenic differentiation. The involvement of TNF-α and IL-10 in the regulation of MSC functions was investigated using neutralizing antibodies and recombinant cytokines.

Results: Treatment of MSC with media conditioned by pro-inflammatory or anti-inflammatory macrophages promoted cell elongation and enhanced MSC ability to attach and migrate. These effects were more noticeable when MSC were treated with media from pro-inflammatory macrophages. Interestingly, MSC osteogenic activity was enhanced by factors released by anti-inflammatory macrophages, but not by pro-inflammatory macrophages. Significant IL-10 levels originated from anti-inflammatory macrophages enhanced MSC osteogenesis by increasing ALP activity and mineralization in MSC layers cultured under osteogenic conditions. Moreover, macrophage-derived IL-10 regulated the expression of the osteogenic markers RUNX2, COL1A1, and ALPL. Notably, low TNF-α levels secreted by anti-inflammatory macrophages increased ALP activity in differentiating MSC whereas high TNF-α levels produced by pro-inflammatory macrophages had no effects on osteogenesis. Experiments in which MSC were treated with cytokines revealed that IL-10 was more effective in promoting matrix maturation and mineralization than TNF-α.

Conclusions: Factors secreted by pro-inflammatory macrophages substantially increased MSC attachment and migration whereas those released by anti-inflammatory macrophages enhanced MSC osteogenic activity as well as cell migration. IL-10 was identified as an important cytokine secreted by anti-inflammatory macrophages that potentiates MSC osteogenesis. Our findings provide novel insights into how environments provided by macrophages regulate MSC osteogenesis, which may be helpful to develop strategies to enhance bone regeneration.