Effect of primary osteoblast-derived extracellular vesicles on osteoclast differentiation
10.3724/zdxbyxb-2024-0148
- VernacularTitle:原代成骨细胞来源胞外囊泡促进破骨细胞分化
- Author:
Lan ZHANG
1
;
Jingyi TAN
Author Information
1. 浙江医院口腔科,浙江 杭州 310030
- Keywords:
Osteoblast;
Extracellular vesicles;
Osteoclast;
Differentiation;
Mice
- From:
Journal of Zhejiang University. Medical sciences
2024;53(4):434-442
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of osteoblast-derived extracellular vesicles(OB-EVs)on the proliferation and differentiation of osteoclasts,and to explore the possible molecular mechanism of extracellular vesicles involved in the communication between osteoblasts and osteoclasts.Methods:Primary osteoblasts were isolated from newborn mouse calvarial bone and induced by β-glycero phosphate,ascorbic acid and dexamethasone.Osteogenic feature was tested by alkaline phosphatase(ALP)and alizarin red S staining.Extracellular vesicles were isolated by ultracentrifugation from the cell culture supernatant.Vesicle morphology was observed by transmission electron microscopy,and the characteristic markers of tumor susceptibility gene 101(TSG101),ALG-2 interacting protein X(Alix)and cluster of differentiation 9(CD9)on the surface of extracellular vesicles were identified by Western blotting.Cell counting kit 8(CCK-8)assay was used to determine the proliferation effect of OB-EVs on mouse mononuclear macrophage RAW264.7 cells.Furthermore,the expression level of specific markers of osteoclast differentiation in RAW264.7 cells was detected by Western blotting after the combined effect of OB-EVs and receptor activator for nuclear factor κB ligand(RANKL).The number of osteoclasts was observed and compared with OB-EVs-treated mouse bone marrow-derived macrophages(BMMs)by tartrate-resistant acid phosphatase(TRAP)staining,and the effect of OB-EVs on osteoclast differentiation was determined.Results:The extracted OB-EVs showed a double-layer cup-like structure with a diameter of 30-150 nm,and TSG101,Alix and CD9 were expressed.RAW264.7 cells were stimulated with OB-EVs,and the results of CCK-8 assay showed that high concentration of OB-EVs(more than 20 μg/mL)inhibited cell proliferation(P<0.05).Western blotting analysis showed that the expression of osteoclast differentiation marker proteins such as c-Fos,activated T cell nuclear factor(NFATc1)and c-Jun N-terminal kinase(JNK)in RAW264.7 cells were significantly increased,and the promoting effect was enhanced with increasing of OB-EVs concentration(P<0.05).In addition,the combination of OB-EVs and RANKL on BMMs showed that the number of TRAP-positive cells was significantly higher than that of the RANKL induction group alone(P<0.05).Conclusion:OB-EVs can promote the differentiation of osteoclast precursor cells into osteoclasts,but high concentration of OB-EVs can inhibit proliferation of RAW264.7 cells.
