Immunomodulatory Peptide Mediates Macrophage Exosome Regulate Bone Regeneration Through MiR-21b

Purpose: Bone defects,especially large areas of bone defects in the oral and maxillofacial region,greatly affect the physical and mental health of patients.However,the existing treatment methods have problems such as insufficient donor sources,poor biocompatibility,and many complications.Therefore,it is urgent to find a "cell-free therapy" that has low immunogenicity and can quickly induce bone tissue regeneration.Exosomes are a class of extracellular vesicles with a diameter of 30-120 nm,and their contents include proteins,lipids,and nucleic acids,which play an important role in intercellular communication.Macrophage exosomes have attracted widespread attention due to their biological properties similar to those of parent cells,and the contents of macrophage exosomes are closely related to environmental stimuli.This study intends to use the immunomodulatory peptide nanomicelle DP7-C,which has dual functions of carrier and immune adjuvant,to stimulate macrophages,and to explore the changes in the content of exosomes derived from it and the mechanism of bone formation.Provide new ideas for bone tissue regeneration therapy.Methods: Exosomes from macrophages were extracted by ultracentrifugation,identified by TEM,NTA,and WB,and the uptake of exosomes by cells was observed by confocal microscopy;osteogenesis was detected at the gene and protein levels by q PCR and WB The expression of factors was evaluated by ALP and ARS staining to evaluate the effect of exosomes on BMSCs osteogenic efficiency;the composition and difference of mi RNA in exosomes in the blank group and the experimental group were detected by exosomal mi RNA-seq,and bioinformatics analysis was used The target genes and pathways were enriched,and then WB was used to detect the expression of upstream and downstream molecules of the pathway,and dual luciferase was used for target verification;finally,a mouse model of experimental periodontitis was established,and Micro-CT,HE staining,immunohistochemistry Staining to evaluate the effect of exosomes on periodontal destruction in mice.Results: Macrophage exosomes have a typical "saucer"-shaped vesicle-like structure with a particle size between 50-120 nm,express positive marker proteins CD63,CD81,and TSG101,and do not express negative protein Calnexin,and can be phagocytized by BMSCs The exosomes secreted by macrophages stimulated by the immunomodulatory peptide DP7-C can promote the osteogenic differentiation of BMSCs.The exosome mi RNA-seq results showed that there were mi RNAs differentially expressed between them and natural exosomes,among which mi R-21 b was significantly High expression;DP7-C can successfully carry mi R-21 b into macrophages and their secreted exosomes,mi R-21 b in exosomes can promote osteogenesis by targeting SOCS1 degradation and activating JAK2/STAT3 signaling pathway differentiation.Conclusion: The immunomodulatory peptide DP7-C,which has dual functions of carrier and immune adjuvant,can secrete a large amount of exosomes after stimulating macrophages,and its mi RNA composition is different from that of natural macrophage exosomes.Promotes osteogenic differentiation of bone marrow mesenchymal stem cells to the SOCS1/JAK2/STAT3 signaling axis.