Effects Of Macrophage Derived Exosomes Stimulated By Zinc Ions On Endothelial And Osteoblast Functions

Osteogenesis and angiogenesis are both important for implant osseointegration,which can be tailored by immunomodulation of macrophages.Pure zinc(Zn)and its alloys,a novel biodegradable material,can modulate macrophage functions in its ionic form.However,whether macrophage-derived exosomes,novel carriers of intracellular communication,participate in the process is still unclear.Therefore,in this study,macrophages were cultured in the medium containing different concentrations of Zinc ions,and macrophage derived exosomes were successfully extracted by ultracentrifugation.Then the exosomes were characterized and analyzed by scanning electron microscopy and Western blot.Finally,different exosomes were extracted to act on endothelial cells and osteoblasts,and evaluated the effect of macrophage-derived exosomes on the biocompatibility,angiogenesis,and osteogenic differentiation.The main research results are as follow:(1)Low concentration of zinc ions can promote the proliferation of macrophages,but the difference between the groups is not obvious.When the zinc ion concentration is above500μM,it shows cytotoxicity and the cells cannot survive.At the same time,different zinc ion concentrations have no significant effect on the morphology of macrophages,but will down-regulate the M1 and M2 genes of macrophages.(2)The presence of exosomes was confirmed by SEM and western blot,exosomes were spherical under SEM and the particle size is mainly in the range of 30-150 nm.The results of EDS about exosomes showed that Zinc ions had no significant effect on the amount of exosomes secreted by macrophages,but will make the zinc content in exosomes has slightly increased.(3)After the exosomes were taken up by endothelial cells,the normal macrophagederived exosomes could negatively regulate the angiogenic ability of endothelial cells,while the exosomes of macrophages stimulated by zinc ions could positively regulate their angiogenic ability.Exosomes can promote the adhesion of endothelial cells,NO synthesis and VEGF secretion and the Exo-Zn20 group was the best.(4)Macrophage-derived exosomes can promote the cell viability of osteoblasts.Macrophage-derived exosomes can promote ALP activity in the early stage of osteogenic differentiation and it can promote the secretion of Ⅰ collagen of osteoblasts.And the Exo-Zn4 group was obvious.The present work shows,for the first time,the exosomes derived from macrophages upon Zn ion stimulation can promote endotheliocytes migration,a key step of angiogenesis,and upregulate osteoblast ALP activity and promote the secretion of Ⅰ collagen,an early marker of osteogenic differentiation,thus enriching the immunomodulatory mechanisms of macrophages.