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-1ß, 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.

Fibrotic alterations in human annulus fibrosus correlate with progression of intervertebral disc herniation.

BACKGROUND: Intervertebral disc (IVD) herniation is characterized by annulus fibrosus failure (AF) in containing the nucleus pulposus (NP). IVD herniation involves cellular and extracellular matrix (ECM) alterations that have been associated with tissue fibrosis, although still poorly investigated. METHODS: Here, fibrotic alterations in human AF were evaluated, by characterizing the herniated ECM. Human AF samples (herniated lumbar IVD (n = 39, age 24-83) and scoliosis controls (n = 6, age 15-21)) were processed for transmission electron microscopy and histological/immunohistochemical analysis of fibrotic markers. Correlations between the fibrotic markers in AF ECM and the degree of NP containment (protused, contained and uncontained) and patients' age were conducted. RESULTS: Our results demonstrate that with herniation progression, i.e. loss of NP containment, human AF presents less stained area of sulphated glycosaminoglycans and collagen I, being collagen I fibres thinner and disorganized. On the other hand, fibronectin stained area and percentage of α-smooth muscle actin+ cells increase in human AF, while matrix metalloproteinase-12 (MMP12) production and percentage of macrophages (CD68+ cells) remain constant. These structural and biochemical fibrotic alterations observed in human AF with herniation progression occur independently of the age. CONCLUSIONS: The characterization of human AF here conducted evidence the presence of fibrosis in degenerated IVD, while highlighting the importance of considering the herniation progression stage, despite the patients' age, for a better understanding of the mechanisms behind AF failure and IVD herniation.

IL-1ß-pre-conditioned mesenchymal stem/stromal cells' secretome modulates the inflammatory response and aggrecan deposition in intervertebral disc.

Mesenchymal stem/stromal cells (MSCs) have been increasingly used in clinical trials for low-back pain (LBP) and intervertebral disc (IVD) degeneration with promising results. Their action mechanisms are not fully understood, but they reduce IVD pro-inflammatory markers in a pro-inflammatory/degenerative IVD microenvironment. In this study the therapeutic potential of the MSC secretome, as an alternative cell-free approach for treating degenerated IVDs, was examined. Human bone marrow-derived MSC secretome (MSCsec) was collected after 48 h of preconditioning in IL-1ß (10 ng/mL) and low oxygen (6 % O2), mimicking the degenerative IVD. IL-1ß-pre-conditioning of MSCs increased secretion of pro-inflammatory markers hIL-6, hIL-8, hMCP-1, etc. The therapeutic effect of MSCsec was tested in a pro-inflammatory/degenerative IVD ex vivo model. MSCsec down-regulated IVD gene expression of pro-inflammatory cytokines (bIL-6, bIL-8) and matrix degrading enzyme bMMP1, while bMMP3 and bTIMP2 were up-regulated, at 48 h. After 14 d, MSCsec-treated IVDs revealed increased aggrecan deposition, although no differences in other ECM components were observed. Protein analysis of the MSCsec-treated IVD supernatant revealed a significant increase of CXCL1, MCP-1, MIP-3α, IL-6, IL-8 and GRO α/ß/γ (related to TNF, NOD-like receptor and neutrophil chemotaxis signalling), and a decrease of IFN-γ, IL-10, IL-4, IL-5 and TNF-α (associated with T-cell receptor signalling). MSCsec-treated IVD supernatants did not promote angiogenesis and neurogenesis in vitro. Overall, MSCsec can be a safe therapeutic approach, presenting a strong immunomodulatory role in degenerated IVD while potentiating aggrecan deposition, which can open new perspectives on the use of MSCsec as a cell-based/ cell-free therapeutic approach to LBP.