Effects of magnesium-based materials on immune response mediated osteogenesis

Background: Human mesenchymal stem cells (MSC) interact with numerous immune cells that can promote regenerative processes and inhibit inflammatory responses. It is hypothesised that the crosstalk between human umbilical cord perivascular cells (HUCPV; an alternative source of MSC) and peripheral blood mononuclear cells (PBMC)/macrophages could be influenced by degradable magnesium (Mg) and its alloy (Mg-10Gd; 10 weight % gadolinium). Moreover, the mechanisms of Mg-based material-modulated immune reactions—macrophage functions and subsequent bone formation—still need to be investigated. Methods: For analysis of the correlations between paracrine signalling and specific cellular behaviour during the initial host response to Mg, two in vitro indirect coculture systems of HUCPV and PBMC were developed in 7 days: (i) transwell (TW) and (ii) conditioned media (CM). In the subsequent 7-14 days, for analysis of the roles of Mg/Mg-10Gd on secretions of macrophages and their effects on pro-osteogenic activity, a direct coculture system of HUCPV and macrophages was established. Results: Cell growth was not significantly influenced by Mg (TW, CM and direct coculture system) or Mg-10Gd (direct coculture system). In the TW system, when HUCPV were cultured with degradable Mg, moderate inflammation (i.e., decreased secretion of proinflammatory interleukin 1 beta (IL1β), IL2, tumour necrosis factor alpha (TNFα), and interferon gamma (IFNγ) and anti-inflammatory IL4, IL5, IL10, IL13, and 1 receptor antagonist (IL1RA) and granulocyte colony stimulating factor (G-CSF)), as well as an increased pro-healing M2 macrophage phenotype, was observed. Moreover, when PBMC were cultured with degradable Mg, the expression of migration/wound healing-related cytokines (IL8, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein (MIP)-1α/β) increased, accompanied by an increased migration of HUCPV (cell scratch assay). In addition, an increased pro-osteogenic potential was demonstrated via an increase in osteoblastic markers (e.g., alkaline phosphatase (ALP) activity, specific gene expression and cytokine release). In the CM system, the percentage of the M2 macrophage phenotype could only be increased by HUCPV and/or Mg. In the direct coculture system, Mg and Mg-10Gd were found to modulate osteogenic differentiation through oncostatin M (OSM) and glycoprotein 130 (gp130). Furthermore, both materials upregulated the gene expression of bone morphogenetic protein 6 (BMP6) in macrophages and of bone morphogenetic protein receptor type 1A and 2 (BMPR1A/2) and mothers against decapentaplegic homologue (Smad) 1/4/5 in cocultured MSC. In addition, both treatments could reduce the secretion of TNFα and IL1β in macrophages and cocultures, which might act synergistically in providing a pro-osteogenic microenvironment. Conclusion: These results collectively imply that Mg possesses osteoimmunomodulatory properties. This study also highlights the roles of material-activated macrophages in pro-osteogenic activity via the OSM/gp130 and Smad-related signalling pathways. Moreover, these findings provide insight into developing Mgbased bone substitute biomaterials capable of desired immune reactions (e.g., tissue regenerative M2 macrophage phenotype) and therapeutic applications using interactions of immune cells and MSC for bone regeneration.


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