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

References 53

1	CHEATHAM S W, HANNEY W, KOLBER M, et al. Osteoporosis: exercise programming insight for the sports medicine professional[J]. Strength Cond J, 2017, 39: 2-13.
2	SI L, WINZENBERG T M, JIANG Q, et al. Projection of osteoporosis-related fractures and costs in China: 2010‒2050[J]. Osteoporos Int, 2015, 26(7): 1929-1937.
3	YU F, XIA W B. The epidemiology of osteoporosis, associated fragility fractures, and management gap in China[J]. Arch Osteoporos, 2019, 14(1): 32.
4	LEE C W, LIN H C, WANG B Y, et al. Ginkgolide B monotherapy reverses osteoporosis by regulating oxidative stress-mediated bone homeostasis[J]. Free Radic Biol Med, 2021, 168: 234-246.
5	LIU P, LEE S, KNOLL J, et al. Loss of menin in osteoblast lineage affects osteocyte-osteoclast crosstalk causing osteoporosis[J]. Cell Death Differ, 2017, 24(4): 672-682.
6	LEE K M, LEE C Y, ZHANG G, et al. Methylglyoxal activates osteoclasts through JNK pathway leading to osteoporosis[J]. Chem Biol Interact, 2019, 308: 147-154.
7	BARSONY J, XU Q, VERBALIS J G. Hyponatremia elicits gene expression changes driving osteoclast differentiation and functions[J]. Mol Cell Endocrinol, 2022, 554: 111724.
8	JACOME-GALARZA C E, PERCIN G I, MULLER J T, et al. Developmental origin, functional maintenance and genetic rescue of osteoclasts[J]. Nature, 2019, 568(7753): 541-545.
9	PESCE VIGLIETTI A I, GIAMBARTOLOMEI G H, DELPINO M V. Endocrine modulation of Brucella abortus-infected osteocytes function and osteoclastogenesis via modulation of RANKL/OPG[J]. Microbes Infect, 2019, 21(7): 287-295.
10	KIM J M, LIN C J, STAVRE Z, et al. Osteoblast-osteoclast communication and bone homeostasis[J]. Cells, 2020, 9(9): 2073.
11	QIAN J, HE Y, ZHAO J, et al. IL4/IL4R signaling promotes the osteolysis in metastatic bone of CRC through regulating the proliferation of osteoclast precursors[J]. Mol Med, 2021, 27(1): 152.
12	YAO Z, GETTING S J, LOCKE I C. Regulation of TNF-induced osteoclast differentiation[J]. Cells, 2021, 11(1): 132.
13	KANG M Y, HUANG C C, LU Y, et al. Bone regeneration is mediated by macrophage extracellular vesicles[J]. Bone, 2020, 141: 115627.
14	CAI F Y, LIU S L, LEI Y X, et al. Epigallocatechin-3 gallate regulates macrophage subtypes and immunometabolism to ameliorate experimental autoimmune encephalomyelitis[J]. Cell Immunol, 2021, 368: 104421.
15	ZHANG Z G, ZHANG C Y, ZHANG S R. Irisin activates M1 macrophage and suppresses Th2-type immune response in rats with pelvic inflammatory disease[J]. Evid Based Complement Alternat Med, 2022, 2022: 5215915.
16	EOM J, YOO J, KIM J J, et al. Viperin deficiency promotes polarization of macrophages and secretion of M1 and M2 cytokines[J]. Immune Netw, 2018, 18(4): e32.
17	ZHANG W J, GUAN N, ZHANG X M, et al. Study on the imbalance of M1/M2 macrophage polarization in severe chronic periodontitis[J]. Technol Health Care, 2023, 31(1): 117-124.
18	WANG W H, LIU H, LIU T, et al. Insights into the role of macrophage polarization in the pathogenesis of osteoporosis[J]. Oxid Med Cell Longev, 2022, 2022: 2485959.
19	YU L, HU M, CUI X, et al. M1 macrophage-derived exosomes aggravate bone loss in postmenopausal osteoporosis via a microRNA-98/DUSP1/JNK axis[J]. Cell Biol Int, 2021, 45(12): 2452-2463.
20	LU Y P, LIU S S, YANG P P, et al. Exendin-4 and eldecalcitol synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells through M2 macrophages polarization via PI3K/AKT pathway[J]. Stem Cell Res Ther, 2022, 13(1): 113.
21	CHEN M, LIN W M, YE R, et al. PPARβ/δ agonist alleviates diabetic osteoporosis via regulating M1/M2 macrophage polarization[J]. Front Cell Dev Biol, 2021, 9: 753194.
22	WEI H, CHEN Q, LIN L, et al. Regulation of exosome production and cargo sorting[J]. Int J Biol Sci, 2021, 17(1): 163-177.
23	LI M D, JIA J, LI S S, et al. Exosomes derived from tendon stem cells promote cell proliferation and migration through the TGF β signal pathway[J]. Biochem Biophys Res Commun, 2021, 536: 88-94.
24	WANG S W, JU T Y, WANG J J, et al. Migration of BEAS-2B cells enhanced by H1299 cell derived-exosomes[J]. Micron, 2021, 143: 103001.
25	SHARIATI NAJAFABADI S, AMIRPOUR N, AMINI S, et al. Human adipose derived stem cell exosomes enhance the neural differentiation of PC12 cells[J]. Mol Biol Rep, 2021, 48(6): 5033-5043.
26	YANG S D, GUO S, TONG S, et al. Promoting osteogenic differentiation of human adipose-derived stem cells by altering the expression of exosomal miRNA[J]. Stem Cells Int, 2019, 2019: 1351860.
27	ZHANG B B, ZHAXI D W, LI C, et al. M2 macrophagy-derived exosomal miRNA-26a-5p induces osteogenic differentiation of bone mesenchymal stem cells[J]. J Orthop Surg Res, 2022, 17(1): 137.
28	WEN X, HU G, XIAO X, et al. FGF2 positively regulates osteoclastogenesis via activating the ERK-CREB pathway[J]. Arch Biochem Biophys, 2022, 727: 109348.
29	ZHU G C, CHEN W, TANG C Y, et al. Knockout and double knockout of cathepsin K and Mmp9 reveals a novel function of cathepsin K as a regulator of osteoclast gene expression and bone homeostasis[J]. Int J Biol Sci, 2022, 18(14): 5522-5538.
30	HE F T, LUO S H, LIU S J, et al. Zanthoxylum bungeanum seed oil inhibits RANKL-induced osteoclastogenesis by suppressing ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest in RAW264.7 cells[J]. J Ethnopharmacol, 2022, 289: 115094.
31	KUMAR A, HUGHES T M, CRAFT S, et al. A novel approach to isolate brain-cell-derived exosomes from plasma to better understand pathogenesis of Alzheimer's disease[J]. Alzheimer's Dement, 2020, 16(Suppl 4): e044894.
32	LI K, WONG D K, HONG K Y, et al. Cushioned-density gradient ultracentrifugation (C-DGUC): a refined and high performance method for the isolation, characterization, and use of exosomes[J]. Methods Mol Biol, 2018, 1740: 69-83.
33	HELWA I, CAI J W, DREWRY M D, et al. A comparative study of serum exosome isolation using differential ultracentrifugation and three commercial reagents[J]. PLoS One, 2017, 12(1): e0170628.
34	DING M, WANG C, LU X L, et al. Comparison of commercial exosome isolation kits for circulating exosomal microRNA profiling[J]. Anal Bioanal Chem, 2018, 410(16): 3805-3814.
35	LIANG B, BURLEY G, LIN S, et al. Osteoporosis pathogenesis and treatment: existing and emerging avenues[J]. Cell Mol Biol Lett, 2022, 27(1): 72.
36	LI K, XIU C M, ZHOU Q, et al. A dual role of cholesterol in osteogenic differentiation of bone marrow stromal cells[J]. J Cell Physiol, 2019, 234(3): 2058-2066.
37	CHE Y T, YANG J Z, TANG F, et al. New function of cholesterol oxidation products involved in osteoporosis pathogenesis[J]. Int J Mol Sci, 2022, 23(4): 2020.
38	LI K Q, CHEN S H, CAI P Y, et al. MiRNA-483-5p is involved in the pathogenesis of osteoporosis by promoting osteoclast differentiation[J]. Mol Cell Probes, 2020, 49: 101479.
39	PARK E, LEE C G, LIM E, et al. Osteoprotective effects of loganic acid on osteoblastic and osteoclastic cells and osteoporosis-induced mice[J]. Int J Mol Sci, 2020, 22(1): 233.
40	LAI G H, ZHAO R L, ZHUANG W D, et al. BMSC-derived exosomal miR-27a-3p and miR-196b-5p regulate bone remodeling in ovariectomized rats[J]. PeerJ, 2022, 10: e13744.
41	SONG H Y, LI X Q, ZHAO Z C, et al. Reversal of osteoporotic activity by endothelial cell-secreted bone targeting and biocompatible exosomes[J]. Nano Lett, 2019, 19(5): 3040-3048.
42	CHEN X T, WAN Z, YANG L, et al. Exosomes derived from reparative M2-like macrophages prevent bone loss in murine periodontitis models via IL-10 mRNA[J]. J Nanobiotechnology, 2022, 20(1): 110.
43	ZHU L F, LI L, WANG X Q, et al. M1 macrophages regulate TLR4/AP1 via paracrine to promote alveolar bone destruction in periodontitis[J]. Oral Dis, 2019, 25(8): 1972-1982.
44	LIANG B L, WANG H C, WU D, et al. Macrophage M1/M2 polarization dynamically adapts to changes in microenvironment and modulates alveolar bone remodeling after dental implantation[J]. J Leukoc Biol, 2021, 110(3): 433-447.
45	SHI M S, WANG C, WANG Y L, et al. Deproteinized bovine bone matrix induces osteoblast differentiation via macrophage polarization[J]. J Biomed Mater Res A, 2018, 106(5): 1236-1246.
46	SHI C, YUAN F, LI Z L, et al. MSN@IL-4 sustainingly mediates macrophagocyte M2 polarization and relieves osteoblast damage via NF-κB pathway-associated apoptosis[J]. Biomed Res Int, 2022, 2022: 2898729.
47	Horibe K, Hara M, Nakamura H. M2-like macrophage infiltration and transforming growth factor-β secretion during socket healing process in mice[J]. Arch Oral Biol, 2021, 123: 105042.
48	WANG X Y, JI Q B, HU W H, et al. Isobavachalcone prevents osteoporosis by suppressing activation of ERK and NF-κB pathways and M1 polarization of macrophages[J]. Int Immunopharmacol, 2021, 94: 107370.
49	LI Z K, ZHU X D, XU R J, et al. Deacylcynaropicrin inhibits RANKL-induced osteoclastogenesis by inhibiting NF-κB and MAPK and promoting M2 polarization of macrophages[J]. Front Pharmacol, 2019, 10: 599.
50	YAO M Y, CUI B, ZHANG W H, et al. Exosomal miR-21 secreted by IL-1β-primed-mesenchymal stem cells induces macrophage M2 polarization and ameliorates sepsis[J]. Life Sci, 2021, 264: 118658.
51	MA J, CHEN L, ZHU X, et al. Mesenchymal stem cell-derived exosomal miR-21a-5p promotes M2 macrophage polarization and reduces macrophage infiltration to attenuate atherosclerosis[J]. Acta Biochim Biophys Sin (Shanghai), 2021, 53(9): 1227-1236.
52	LI R, LI D Z, WANG H N, et al. Exosomes from adipose-derived stem cells regulate M1/M2 macrophage phenotypic polarization to promote bone healing via miR-451a/MIF[J]. Stem Cell Res Ther, 2022, 13(1): 149.
53	LI R, ZHAO K C, RUAN Q, et al. Bone marrow mesenchymal stem cell-derived exosomal microRNA-124-3p attenuates neurological damage in spinal cord ischemia-reperfusion injury by downregulating Ern1 and promoting M2 macrophage polarization[J]. Arthritis Res Ther, 2020, 22(1): 75.

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