Abstract
Background: Human mesenchymal stem cells (MSC) interact with numerous immune cells that can promote regenerative processes and inhibit inflammatory responses.We hypothesised that the cross-talk 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 materials modulated immune reaction – the macrophage functions, and subsequent bone formation, are still need to be further investigated. Methods: Within 7 days, to study the correlations between paracrine signalling and specific cellular behaviour during initial host response against to Mg, two in vitro indirect coculture systems of HUCPV and PBMC were developed: (i) transwell (TW) and (ii) conditioned media (CM). In the subsequent 7-14 days, to analysis 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) and Mg-10Gd (direct coculture system). In TW system, when HUCPV were cultured with degradable Mg, a moderate inflammation (e.g., lower secretions of pro-inflammatory interleukin 1 beta, 2, and tumour necrosis factor alpha, interferon gamma, and anti-inflammatory interleukins 4, 5, 10, 13, and 1 receptor antagonist and granulocyte colony stimulating factor), as well as an increased pro-healing M2 macrophages phenotype, were exhibited; Moreover, when PBMC were cultured with degradable Mg, the expression of migration/wound healing related cytokines (interleukin 8, granulocyte-macrophage colony-stimulating factor, monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α/β) raised, accompanying with an increased migration ability of HUCPV (cell scratch assay); In addition, an increased pro-osteogenic potential was demonstrated via an increase of osteoblastic markers (e.g., alkaline phosphatase activity, specific gene expression and cytokines release). In CM system, the percentage of M2 macrophages phenotype could only be increased by HUCPV and/or Mg. In 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 homolog (Smad) 1/4/5 in cocultured MSC; In addition, both materials could reduce the secretion of tumour necrosis factor alpha (TNFα) and interleukin 1 beta (IL1β) in macrophages and cocultures, which might act synergistically in providing a pro-osteogenic microenvironment. Conclusion: These results collectively imply that Mg possesses osteo-immunomodulatory properties. This study also highlights the roles of materials-activated macrophages in pro-osteogenic activity via the OSM/gp130 and SMAD-related signalling pathway. Moreover, these findings provide an insight into developing Mg-based bone substitute biomaterials capable of desired immune reactions (i.e., tissue regenerative M2 macrophages phenotype) and therapeutic applications using interactions of immune cells and MSC for bone regeneration.
