| Background and ObjectiveIn recent years,implant restoration has become the preferred restoration method for patients with dentition defects and missing dentition.Currently,the implant metal material used in oral clinics is mainly titanium.Although it has excellent biocompatibility,implant placement In vivo,in the complex human environment,the surface of the implant will release titanium ions into the surrounding tissues and the micron-sized titanium particles on the surface of the implant can stimulate significant inflammatory responses and reduce the bond between the bone and the implant,which may it is one of the important reasons for aseptic loosening of implants.In recent years,the exploration of the pathological mechanism of poor osseointegration of implants caused by aseptic loosening has always been one of the difficulties and hotspots in scientific research.Therefore,elucidating its pathogenic mechanism will be of great significance to prolong the service life of implant dentures clinically.Macrophages are important cells in the microenvironment of tissue inflammation and are widely involved in the initial stage of inflammation,which may mediate the bone loss around implants caused by titanium particles or ions.Macrophage polarization is involved in the regulation of a variety of biological processes,but the role of macrophages in inhibiting osteoblast activity in the titanium ion environment remains unclear.Then we performed transcriptome sequencing analysis and screened out the gene Agt with high confidence.The encoded product of the gene Agt is angiotensinogen(Agt),which is an α-2-globulin mainly produced by the liver and released into the blood circulation.AGT gene is up-regulated in patients with osteoarthritis and may play a key role in the occurrence and progression of osteoarthritis.However,whether up-regulation or down-regulation of Agt gene can affect the dysfunction of titanium ion on osteoblasts via macrophages and the underlying mechanism has not been reported.In conclusion,this study aims to explore the titanium ions through the effect on osteoblast osteogenetic activity of macrophages,clear macrophage polarization and osteoblast of Agt gene is involved in the macrophage mediated titanium ion damage osteoblast osteogenetic activity in the process,to reveal the titanium ion by macrophages in the implant the function and mechanism of aseptic loosening.Materials and methods1.Effect of Titanium ions on osteogenic ability of osteoblasts via macrophagesThe osteoblasts were selected from the mouse osteoblast cell line MC3T3-E1 cells,and the macrophages were selected from the mouse macrophage cell line RAW264.7 cells.The effects of different concentrations of titanium ions on the proliferation ability of osteoblasts and macrophages were detected by CCK-8,and the cytotoxicity to the two cells was observed,and the concentration of titanium ions that had no effect on cell survival and reproduction was screened out.The experimental group with added titanium ions and the control group without added titanium ions were established,and the osteogenic ability of osteoblasts was observed by staining the osteoblasts with alkaline phosphatase(ALP).The two types of cells were co-cultured in a transwell 6-well plate with a 0.4 μm PET membrane chamber,macrophages were placed on the membrane of the chamber,and osteoblasts were cultured to the bottom of the plate.The experimental group with titanium ions and the control group without titanium ions were established in co-culture mode,and the changes of osteogenic ability were observed under the intervention of different conditions in each group.The expression of osteogenesis-related genes and proteins was mainly detected by ALP staining,qRT-PCR,and Western Blot to reflect the osteogenic ability of the two groups of osteocytes.2.The effect of titanium ions on the polarization of macrophagesAfter co-culture of osteoblasts and macrophages,the macrophages in the titanium ion group were compared with the control group,qRT-PCR,Western Blot,immunofluorescence staining,flow cytometry results showed that Ml-type macrophages increased,M2-type macrophages decreased,and pro-inflammatory cytokines increased.3.Effects of knockdown or overexpression of osteoblast Agt gene on the ability of titanium ion to inhibit osteoblast osteogenesis through macrophagesIn the co-culture mode of osteoblasts and macrophages,the experiment was set up as two groups,the experimental group with titanium ions and the control group without titanium ions.Samples of osteoblasts in the two co-culture systems were collected respectively,and each group was set up with 3 replicate groups.After all samples were collected,transcriptome sequencing analysis was performed by relying on Shanghai Gema Company,mainly including gene quality assessment,transcription factor prediction,database comparison,etc.After obtaining the raw data of gene expression levels for omics analysis,according to the set standard,the up-regulated differential genes between the two groups were determined,and the differential genes were analyzed by gene ontology(GO)and signaling pathways to determine the up-regulated differential genes.Determine the functional enrichment of differential genes and the information on the biological signaling pathways involved.Predict the potential relationship between the reduced osteogenic ability of osteoblasts and a differential gene,and explore the possible mechanism of action.The screened differential genes were verified by qRT-PCR,and the Agt gene with high reliability were found.The osteoblast Agt gene was knocked down and overexpressed by lentiviral transfection,and the knockdown and overexpression efficiencies were detected by Western Blot.After confirming that Agt can be stably knocked down and overexpressed in osteoblasts,a normal medium group,10 μg/mL Ti ion medium group,10 μg/mL Ti+shNC group(knockdown control group),10μg/mL Ti+shAgt group(knockdown experimental group),10 μg/mL Ti+OENC group(overexpression control group),10 μg/mL Ti+OEAgt group(overexpression experimental group)were established in the co-culture system of osteoblasts and macrophages.These groups were compared on the biological function of osteoblasts.ALP staining and Western Blot were mainly used to detect the expression levels of osteogenic-related proteins to detect the osteogenic ability of osteoblasts,and to verify the function of knockdown and overexpression of osteoblast Agt on macrophages in the titanium ion environment.Result1.10 μg/mL titanium ion inhibited the osteogenic ability of osteoblasts through macrophagesThe results of CCK-8 showed that 10 μg/mL titanium ion had no significant effect on the proliferation of osteoblasts and macrophages.ALP staining and Western Blot showed that 10μg/mL titanium ion had no significant effect on the osteogenic ability of osteoblasts.The co-culture model of osteoblasts and macrophages was successfully constructed in vitro.ALP staining,qRT-PCR and Western Blot results showed that compared with the control group,the osteogenic activity of osteoblasts in the macrophage-mediated titanium ion group was reduced,titanium ions may affect the osteogenic ability of osteoblasts through macrophages.2.10 μg/mL titanium ions produce inflammatory responses by regulating the polarization of macrophages towards M1After co-culture of osteoblasts and macrophages,the macrophages in the titanium ion group were compared with the control group,qRT-PCR,Western Blot,immunofluorescence cell staining,flow cytometry results showed that M1 macrophages increased,M2 macrophages reduced and pro-inflammatory cytokines increased.3.Knockdown of osteoblast Agt gene improved the reduction of osteoblast osteogenic activity mediated by macrophage by 10μg/mL titanium ion,while overexpression of osteoblast Agt gene was reversed Through RNA-seq,the gene differences between the two groups of osteoblasts with and without titanium ions in the co-culture system of osteoblasts and macrophages were compared,and based on P value<0.05 and absolute fold change≥1.2 basic principles were used to screen genes,and a total of 63 differential genes were obtained.The results of bioinformatics analysis indicated that the differential genes were mainly enriched in the extracellular matrix,biological regulation,and ion binding in terms of GO function,and were mainly enriched in related biological processes such as inflammation and bone metabolism in signaling pathways.The Agt gene knockdown and overexpression models of osteoblasts were successfully constructed by lentivirus transfection in vitro with good transfection efficiency.When examining the osteogenic function of each group of cells,it was found that knocking down Agt gene could improve the inhibiting effect of titanium ion on osteoblast osteogenic function mediated by macrophages,while overexpression of Agt gene could aggravate the inhibiting effect of osteoblast osteogenic function.Conclusions1.10 μg/mL titanium ion inhibited the osteogenic function of osteoblasts through macrophages.2.Macrophages polarized to M1 type and produced inflammatory response in 10 μg/mL titanium ion environment.3.By knocking down the Agt gene of osteoblasts,the damage of osteoblasts induced by 10 μg/mL titanium ion mediated by macrophages was ameliorated. |
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