Effect Of CPC/PLGA On MiRNA Expression Of Exosomes During Osteoclastic Differentiation Of Monocytes

Objective: The purpose of this study is to investigate the effects of CPC/PLGA degradation products on the osteoclast differentiation of monocytes and the changes in the expression profile of exosome mi RNAs secreted during osteoclast differentiation,and to provide theoretical basis for the promotion of new bone formation in the area where the material is implanted.Experiment 1 Effect of CPC/PLGA degradation products on osteoclastic differentiation of monocytesMethods: The materials after CPC/PLGA degradation were simulated with a compound degradation solution.Receptor Activator for Nuclear Factor-κ B Ligand(RANKL)was added into the culture medium at a concentration of50ng/m L.RAW264.7 cells were induced into osteoclast-like cells in five days.CCK-8 method was used to analyze the effects of different media on the proliferation of RAW264.7 cells.The expression level of m RNA and protein of NFATC1,MMP-9,RANK and TRAP was detected by RT-PCR and WB.The formed osteoclasts were stained with tartrate-resistant acid phosphatase staining(TRAP)reagent.Results: The CCK-8 results showed that the cell proliferation rate of each group was faster in the first three days,and the number of cells reached the peak on the fourth day and then decreased;RT-PCR and WB results showed that the m RNA expression level of NFATC1,RANK,TRAP and the protein expression level of NFATC1,RANK in CPC/PLGA group were significantly higher than the GA group and control group.There was no significant difference in the m RNA and protein expression levels of MMP-9 between CPC/PLGA groups in the GA group,both of which were higher than the control group.TRAP staining showed that the number of osteoclast like cells in CPC/PLGA group was more than that in the GA and control group.Experiment 2 The effect of CPC/PLGA degradation products on mi RNA expression profile of exosomes derived from monocytesMethods: According to the results of Experiment 1,exosomes were extracted from the supernatant of cells in the control group and CPC/PLGA group,which were cultured for 5 days in the medium supplemented with50ng/ml RANKL.The extracted exosome mi RNAs were sequenced,the mi RNAs with significant differences were screened and the corresponding target genes were searched,and the target genes of differentially expressed mi RNAs were enriched by GO analysis and KEGG pathway enrichment analysis.Results: Among the mi RNAs with significant difference between CPC/PLGA group and control group,77 mi RNAs with significant difference were screened out,of which 27 were up-regulated and 50 were down-regulated.Among them,mi RNAs involved in the process of osteoclastic differentiation of monocytes included mi R-3090-5p,mi R-346-3p,mi R-714,mi R-706 and mi R-3473b;GO enrichment analysis showed that the target genes were concentrated in the processes related to apoptosis and cell metabolism mainly.KEGG analysis showed that the signaling pathways related to the differentiation of monocytes into osteoclasts included p53 signaling pathway,PI3K/AKT signaling pathway,TNF signaling pathway,NF-κB signaling pathway.Conclusions: Under the conditions of this experiment,the materials after CPC/PLGA degradation can promote osteoclastic differentiation of monocytes and change the expression profile of mi RNA in exosomes derived from monocytes.Then,mi R-3090-5p,mi R-346-3p,mi R-714 Mi R-706 and mi R-3473 b are associated with the process of osteoclast differentiation of monocytes,and may participate in the differentiation process of osteoclasts through the regulation of multiple signaling pathways such as p53,PI3K/AKT,TNF enriched by mi RNA target genes.