The Role Of Magnesium In The Chondrogenic Differentiation Of Mesenchymal Stem Cells Via Mediating Macrophage Polarization

[Objective] Magnesium alloy is regarded as a promising implant in department of orthopedics,accompanying the important immunomodulatory effects of released magnesium ions.In the present study,we aim to detect the potential effects of magnesium sulfate on the polarization of macrophages and their production of inflammatory cytokines with or without the activation of interferon-γ(IFN-γ) and lipopolysaccharide(LPS).We also investigate whether the immunomodulatory effects of magnesium sulfate on macrophages have influence on the chondrogenesis of mesenchymal stem cells(MSCs) and the proliferation and migration of chondrocytes.We hope the results we obtained could provide theoretical basis for the application of magnesium alloy in the future.[Methods] Firstly,LPS and IFN-γ were used to induce macrophages to differentiate into M1 phenotype and interleukin(IL)-4 were used to induce the generation of M2 macrophages.Flow cytometry was applied to detect the effects of different concentrations of magnesium sulfate on the polarization of macrophages during the induction process.Meanwhile,by ELISA and RT-qPCR,we tested the generation of inflammatory cytokines released by induced macrophages with or without the treatment of magnesium sulfate.Then,we collected the supernatants from M1-polarized macrophages with or without magnesium sulfate treatment and mixed them into the cartilage-induced medium to culture human bone MSCs(hBMSCs) in a modified micromass culture system.The obtained micromasses were used to determine the effects of M1-polarized macrophages on the chondrogenesis of hBMSCs by cartilage-related staining,glycosaminoglycans(GAGs)quantification and the expression of chondrogenic genes.Meanwhile,we also tested the effects of magnesium sulfate on the chondrogenesis of hBMSCs by regulating macrophage polarization with the methods mentioned above.In addition,we determined the effects of M1-polarized macrophages on the proliferation and migration of chondrocytes by CCK-8 assay and scratch test.The influence of magnesium sulfate mediated-macrophages polarization on chondrocyte function were also tested accordingly.[Results] Magnesium sulfate of different concentrations did not obviously affect the release of LDH in macrophages but could inhibit the increased release of LDH caused by LPS and IFN-γ.Magnesium sulfate of different concentrations did not notably influence the positive rates of CCR7 and CD206 in macrophages.However,5 mM magnesium sulfate inhibited the activation of macrophages induced by LPS and IFN-γ,with a notable decrease of the CCR7-positive rate in macrophages,while the CD206-positive rate did not change substantially.Meanwhile,magnesium sulfate of different concentrations did not significantly affect the M2 polarization of macrophages induced by IL-4.Accordingly,the production of many inflammatory cytokines released by activated macrophages was inhibited by magnesium sulfate,such as IL-1β,IL-6,IL-10,as well as their expression at mRNA level.Data from the immunofluorescence staining and western blot analysis showed that magnesium sulfate inhibited the M1 polarization of macrophages and accompanying release of inflammatory cytokines by restraining the activation of NF-κB signaling pathway.The obtained maciomasses were tested by histological staining,GAG quantification and RT-qPCR and we found the inflammation caused by M1 macrophages could inhibit the chondrogenesis of hBMSCs.However,magnesium sulfate could mediate the polarization of macrophages and inhibit the release of inflammatory cytokines,indirectly resulting a promotion of the chondrogenesis of hBMSCs by inhibiting the macrophage-induced inflammation.The inflammation caused by M1 macrophages could inhibit the proliferation and migration of chondrocytes,but magnesium sulfate could reverse this trend by inhibiting the inflammation induced by M1 macrophages.[Conclusions] Magnesium sulfate could inhibit the M1 differentiation of macrophages and accompanying release of inflammatory cytokines by restraining the activation of NF-κB signaling pathway.The inhibition of magnesium sulfate on the M1 polarization of macrophages and their release of inflammatory cytokines provides favorable immune environment for the chondrogenic differentiation of hBMSCs,which can also protect the proliferate and migratory ability of chondrocytes.