Ameliorative effects on osteoporosis of small extracellular vesicles derived from bone marrow mesenchymal stem cells

doi:10.3969/j.issn.1674-8115.2023.04.002

Abstract

Abstract:

Objective ·To investigate the effects of small extracellular vesicles (sEVs) derived from human bone marrow mesenchymal stem cells (BMSCs) on the regulation of osteoclast differentiation and macrophage polarization in mice, and mouse model of osteoporosis. Methods ·BMSCs were cultured and sEVs were isolated through differential centrifugation. The isolated sEVs were identified by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). RAW264.7 cells were cultured and stimulated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) to differentiate the cells into osteoclasts. Tartrate-resistant acid phosphatase (TRAP) staining and phalloidin staining were performed to assess the effect of sEVs on osteoclast formation. The expression levels of osteoclast marker genes, i.e., cAMP-response element binding protein (CREB), cathepsin K (CTSK), and Jun proto-oncogene (c-Jun) were examined by real-time quantitative PCR. To polarize RAW264.7 cells to M1 phenotype, they were cultured with lipopolysaccharides; to polarize them to M2 phenotype, they were cultured with interleukin-4 (IL-4) and IL-13. Flow cytometry was performed to detect the effect of sEVs on macrophage polarization. Micro-computed tomography (micro-CT) and TRAP staining were performed to investigate the effect of sEVs on the bone tissues of lumbar vertebrae in osteoporosis mouse models. Results ·TEM and NTA demonstrated that the isolated sEVs had a typical globular structure with a diameter ranging from 30?150 nm. TRAP staining and phalloidin staining showed that BMSC-derived sEVs inhibited the fusion of RAW264.7 cells to form osteoblasts. PCR revealed that sEVs could decrease the expression of CREB, CTSK, and c-Jun (all P<0.05). Flow cytometry analysis indicated that BMSC-derived sEVs inhibited RAW264.7 macrophages polarization to M1 phenotype and induced RAW264.7 macrophages polarization to M2 phenotype. Micro-CT indicated that the number of trabeculae and the bone volume fraction of lumbar vertebrae were significantly higher in the sEV-intervened group than those in the control group (both P<0.05). TRAP staining revealed a reduction of osteoclast number in the lumbar vertebrae after intervention with sEVs. Conclusion ·The sEVs from human BMSCs can delay bone loss in osteoporosis mice, which may be related to its effects of inhibiting osteoclast differentiation and promoting the polarization of M2 type macrophages.

Key words: bone marrow mesenchymal stem cell (BMSC), small extracellular vesicle (sEV), osteoporosis, osteoclast, macrophage polarization

CLC Number:

R681

LI Xuran, TAO Shicong, GUO Shangchun. Ameliorative effects on osteoporosis of small extracellular vesicles derived from bone marrow mesenchymal stem cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(4): 406-416.

Figures/Tables 11

Tab 1 Primer sequences for qPCR

Primer	Sequence
β-actin forward	5'-CCTCTATGCCAACACAGT-3'
β-actin reverse	5'-AGCCACCAATCCACACAG-3'
CREB forward	5'-CCTTGCTTTCCGAATCCTC-3'
CREB reverse	5'-CACTTTGGCTGGACATCTTG-3'
c-Jun forward	5'-AGCAACTTTCCTGACCCAGAG-3'
c-Jun reverse	5'-TCTTTACAGTCTCGGTGGCAG-3'
CTSK forward	5'-CCAGAATCTTGTGGACTGTGT-3'
CTSK reverse	5'-CATCTTCAGAGTCAATGCCTC-3'

Fig 1 Identification of sEVs from BMSCs by TEM and NTA

Fig 2 Formation of osteoclasts and the results of TRAP staining

Fig 3 TRAP staining results of two groups of cells differentiated into osteoclasts (×40)

Fig 4 Phalloidin staining results of two groups of cells differentiated into osteoclasts (×40)

Fig 5 mRNA levels of CREB (A), CTSK (B) and c-Jun (C) detected by qPCR after the cells differentiated into osteoclasts in the two groups

Fig 6 Expression level of M1 macrophage marker (CD86) detected by flow cytometry

Fig 7 Expression level of M2 macrophage marker (CD206) detected by flow cytometry

Fig 8 Observation of sEVs distribution in mouse bones by living imaging

Fig 9 Micro-CT imaging analysis and bone parameters of the osteoporosis mice and the sEV-intervened mice

Fig 10 TRAP staining images of the osteoporosis mice and the sEV-intervened mice

TrendMD

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