| Objective Magnesium and mineralized collagen(n HAC)have become the focus of current research due to their good bone-promoting properties.However,bone tissue growth and repair are not only materials that interact with osteoblasts.Favourable bone integration is always accompanied by rapid blood vessel formation,which provides nutrients for the growth of new bone.In addition,immune responses also play a central regulatory role in the release of pro-inflammatory or anti-inflammatory cytokines and growth factors to manipulate osteogenesis and angiogenesis.The effects of biomaterials on bone immune microenvironment affect the results of bone regeneration.Therefore,Mg2+ and mineralized collagen were used in this study to explore whether magnesium and mineralized collagen can cooperatively regulate the immune response of macrophages and induce the polarization of macrophages to M2 phenotype,and to further study the effect of magnesium and mineralized collagen synergistic effect on the ability of osteogenesis and angiogenesis by regulating immune microenvironment.Methods According to the molecular weight of Mg,S and O,magnesium sulfate was dissolved in complete medium to prepare a 100 mg/L Mg2+ solution.The n HAC were cut with a diameter of 15 mm and a thickness of 2 mm to form n HAC discs.The experimental groups were as follows: blank control group: complete medium;n HAC group: n HAC disc;Mg2+ group: 100mg/L Mg2+;Mg2++n HAC group: 100mg/L Mg2+ +n HAC disc.1.First,CCK-8 was used to detect the proliferation activity of RAW264.7macrophages in the blank control group,n HAC group,Mg2+ group,and Mg2++n HAC group.Then macrophages were induced into M1-type macrophages by LPS,and the blank control group,n HAC group,Mg2+ group,and Mg2++n HAC group were used to culture the LPS-induced RAW264.7macrophages.Flow cytometry was used to evaluate the percentage of M1 macrophages that showed the marker CCR7 and the M2 macrophage surface marker CD206 positive cells;q RT-PCR was used to detect the inflammation-related genes expression of TNF-α,IL-1β,IL-6,IL-10,IL-1ra and the genes expression of cell growth factors BMP-2,PDGF-B.The expression of NF-κB p65 and IκB-α was detected by Western blot.2.RAW264.7 macrophages were cultured in blank control group,n HAC group,Mg2+ group and Mg2++n HAC group respectively.After 2 days,the supernatant of macrophages culture medium was collected and mixed with complete medium in a ratio of 1:2.The resulting medium was RAW conditioned medium.The four groups were denoted as RAW group,n HAC+RAW group,Mg2++RAW group,Mg2+/n HAC+RAW group.Then the four groups of conditioned medium were cultured with MC3T3-E1 and HUVEC cells respectively.ALP activity of MC3T3-E1 cells was detected,mineralization of MC3T3-E1 extracellular matrix was analyzed by alizarin red staining,and osteogenic genes expression of RUNX-2,OPN and OCN were detected by qRT-PCR.In vitro angiogenesis of HUVEC cells was detected by angiogenesis assay,and HIF-1α,KDR and VEGF genes expression were detected by q RT-PCR.Results1.CCK-8 results showed that compared with blank control group,n HAC group,Mg2+ group and Mg2++n HAC group all promoted RAW264.7 macrophage proliferation,and Mg2++n HAC group showed the best cell proliferation activity.After LPS induction of RAW264.7 macrophages,cell phenotype was detected by flow cytometry,which showed that compared with blank control group,the other groups reduced the percentage of CCR7 positive cells of M1 phenotype macrophages,among which Mg2++n HAC group had the lowest percentage of CCR7 positive cells,while the percentage of CD206 positive cells of M2 macrophages in n HAC group,Mg2+ group and Mg2++n HAC group showed an increasing trend,what’s more the percentage of CD206 positive cells in Mg2++n HAC group was the highest.The q RT-PCR results showed that Mg2++n HAC group significantly promoted the expression of macrophage anti-inflammatory gene IL-10 IL-1ra,bone morphogenetic protein 2(BMP-2)and platelets-derived growth factor B(PDGF-B),while significantly inhibited the expression of pro-inflammatory genes TNF-α,IL-1β and IL-6.Western blot analysis showed that compared with blank control group,n HAC group,Mg2+group and Mg2++n HAC group could inhibit expression of NF-κB p65 protein and promote the expression of IκB-α protein,the effect of Mg2++n HAC group was the most significant.2.MC3T3-E1 and HUVEC cells were cultured in RAW cell conditioned medium.Alkaline phosphatase(ALP)activity and alizarin red staining results showed that the ALP activity and mineralization level of MC3T3-E1 cells in Mg2+/n HAC+RAW group were the most obvious,q RT-PCR results showed that the Mg2+/n HAC+RAW group had the most significant osteogenic gene expression level of RUNX-2,OPN and OCN.Meanwhile,the tube formation experiment and q RT-PCR test results showed that compared with other groups,the Mg2+/n HAC+RAW group significantly promoted HUVEC cell angiogenesis and the expression of angiogenesis gene HIF-1α,KDR and VEGF.Conclusions The results of this study showed that Mg2+ combined n HAC significantly reduced the release of inflammatory factors from macrophages,which may be related to the inhibition of NF-κB pathway,and promoted the M2-type polarization of macrophages to generate a favorable bone immune microenvironment to improve osteogenesis and angiogenesis. |
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