Abstract
BackgroundThe 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.MethodsMSC 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.ResultsTreatment 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.ConclusionsFactors 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.
Highlights
Inflammatory response mediated by macrophages plays a vital role in fracture healing [1, 2]
Inflammatory factors secreted by macrophages affect mesenchymal stem cells (MSC) morphology, adhesion, and migration MSC were treated with Conditioned medium (CM) from GM-CSF-generated macrophages (MΦGM) or M-CSF-generated macrophages (MΦM) activated or not with LPS to examine the effects of inflammatory cytokines on MSC (Fig. 1a)
IL-10 and IL-1β levels could not be detected in CM from non-activated MΦGM or MΦM, which contained low concentrations of tumor necrosis factor-alpha (TNF-α), IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) (Table 1)
Summary
Inflammatory response mediated by macrophages plays a vital role in fracture healing [1, 2]. TNF-α, a primary mediator in the inflammatory reaction, promotes the recruitment of inflammatory and stromal cells, stimulates angiogenesis and is essential for bone fracture repair, as demonstrated in TNF-α receptor-deficient mice [3, 4]. In the later stages of inflammation, M2-type macrophages secrete anti-inflammatory cytokines and growth factors, which mediate the resolution of inflammation and tissue repair. The mechanisms by which macrophage phenotype contributes to mesenchymal stem cells (MSC)mediated bone repair remain unclear. 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
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