A Preliminary Study On The Influence Of Mandibular Osteoblasts On The Biological Behavior Of Bone Marrow-derived Macrophages

Macrophages are highly heterogeneous,they can be easily activated under physiological conditions,so there are different subpopulations of macrophages generally exist in the body.Classical activated macrophages(M1)are able to secrete pro-inflammatory cytokines and reactive oxygen species and migrate into inflamed sites as a part of host defenses;alternative activated macrophages(M2)are involved in immune homeostasis by producing anti-inflammatory cytokines and phagocytosing apoptotic cells.Previous studies have confirmed that osteoblasts and osteoclasts both play an important role in the process of bone remodeling,while the immune system have also been found the same effect with the in-depth study by researchers.Therefore,macrophages as one of the important components of the immune system,should not be ignored in the process of bone remodeling.ObjectiveBone marrow-derived macrophages(BMMs)were isolated from rats in vitro to compare the biological behaviors of BMMs that were not activated,IFN-γ and LPS activated,and IL-4 activated.On the basis of this part of the experiment,osteoblasts of the mandible(MOBs)were isolated from rats in vitro and then stimulating BMMs with MOBs conditioned medium to investigate the effects of MOBs on the biological behavior of BMMs.Methods1.Rats BMMs were isolated and cultured in vitro,and identified by morphological observation,Wright Giemsa staining and flow cytometry.2.After being induced and activated by IFN-γ,LPS and IL-4 for 24 h,the morphology of BMMs were observed by inverted microscope,cell surface markers Nos2 and Arg1 were detected by Realtime-PCR.CCK8 method was performed to determine the proliferation ability of BMMs,flow cytometry was performed to detect the apoptosis,and neutral red staining was performed to identify the phagocytic function of BMMs.3.Rats MOBs were isolated and cultured in vitro,and identifying the cells by morphological observation.Alkaline phosphatase(ALP)staining and alizarin red S staining were conducted to detect the osteogenesis capability of MOBs,after culturing them with osteogenic-inducing medium.Then using flow cytometry to identify the CD31 and CD45 as cell surface markers.4.After stimulating BMMs with MOB-conditioned medium(MOB-CM)for 24 h,CCK8 method was used to measure the proliferation ability,flow cytometry was used to detect the apoptosis,neutral red staining was used to identify the phagocytic function,and Realtime-PCR was used to detect the expression of cell surface markers(Nos2,Mrc1),pro-inflammatory related cytokines(Il1b,Il6,Tnfa),anti-inflammatory related factors(Arg1,Il10,Tgfb)and osteoclast differentiation-related genes(Ctsk、Trap、Nfkb1 和 Nfatc1).Results1.BMMs that stimulated by IFN-γ and LPS highly express the M1 cell surface marker Nos2,and can also slightly promote apoptosis and reduce the phagocytic function;while BMMs stimulated by IL-4 highly express the cell surface marker Arg1 of M2,and promote cell proliferation and phagocytosis,significantly inhibit cell apoptosis.2.MOB-CM can inhibit BMMs apoptosis,significantly enhance the phagocytic function of cells,slightly increase the expression of the M1 cell surface marker Nos2,and significantly reduce the expression of Il6 and Tnfa.The expression of osteoclast differentiation-related genes of Ctsk,Trap and Nfatc1 were also down-regulated to varying degrees.Conclusions1.The biological behavior of BMMs can be affected by different methods of polarization induced.2.MOB-CM has a certain effect on the expression of BMMs surface markers and the expression of pro-inflammatory,anti-inflammatory factors and osteoclast differentiation-related factor genes.