Magnesium incorporation in fibrinogen scaffolds promotes macrophage polarization towards M2 phenotype

The host inflammatory response to biomaterials conditions their capacity to promote tissue repair, and macrophage polarization shift from M1 to M2 is determinant in this process. Previous work showed that extracts of a combination between fibrinogen and metallic magnesium materials acted synergistically to reduce macrophage inflammatory phenotype. The hypothesis underlying the current work was that the ability of magnesium-modified fibrinogen scaffolds to modulate macrophage phenotype depends on the concentration of magnesium. Thus, Fibrinogen (Fg) scaffolds incorporating precise concentrations of magnesium sulfate (Mg: 0, 10, 25, 50 mM) were developed and characterized. Mg incorporation in Fg scaffolds increased surface charge, while porosity decreased with increasing Mg concentrations, but only Fg scaffolds with 10 mM of Mg (FgMg10) had significantly improved mechanical properties. Human macrophages cultured on FgMg10 scaffolds, showed increased M2 and decreased M1 polarization, when compared to those cultured on scaffolds with 0, 25 and 50 mM of Mg. Macrophage polarization results were independent of the anion used (chloride or sulfate). Macrophage modulation by FgMg10 scaffolds involved reduced NF-κB p65 nuclear translocation, and impacted production of pro-inflammatory mediators (e.g. IFNγ, IL-12, TNF-⍺, IP-10). Importantly, FgMg10 scaffolds implanted in vivo increased the expression of M2 marker CD163, in macrophages from inflammatory exudates, compared to Sham and Fg-implanted animals, increasing the M2:M1 ratio. A cytokine/chemokine array showed that, while both Fg and FgMg10 scaffolds decreased inflammatory mediators, only FgMg10 decreased IL-1β, IP-10, MIP-2, MDC and MIP-3⍺, compared to Sham-operated animals. This study demonstrated that incorporation of 10mM of Mg modulated inflammation, promoting M2 macrophage polarization in vitro and in vivo. STATEMENT OF SIGNIFICANCE: Developing biomaterials that can modulate inflammation and promote macrophage phenotype switch from M1 to M2 is crucial to promote a regenerative microenvironment. Our previous work showed that extracts of a combination between fibrinogen (Fg) and metallic magnesium (Mg) materials synergistically reduced macrophage pro-inflammatory phenotype. Herein, we tested the hypothesis that macrophage modulation was dependent on Mg concentration. A new family of Fg porous scaffolds incorporating different amounts of Mg (0, 10, 25 and 50 mM) was produced and characterized. We observed that only the combination of Fg scaffolds with 10 mM of Mg (FgMg10) significantly changed the scaffolds mechanical properties and directed macrophages towards a M2 phenotype, reducing the production of inflammatory mediators, both in vitro and in vivo.