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Journal of Peking University (Health Sciences) ›› 2025, Vol. 57 ›› Issue (2): 217-226. doi: 10.19723/j.issn.1671-167X.2025.02.001

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Exosome derived from human adipose-derived mesenchymal stem cells prevented bone loss induced by estrogen deficiency

Chunhui SHENG, Xiao ZHANG, Longwei LV*(), Yongsheng ZHOU*()   

  1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digi-tal Medical Devices & National Health Commission Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2021-11-18 Online:2025-04-18 Published:2025-04-12
  • Contact: Longwei LV, Yongsheng ZHOU E-mail:lvlw@bjmu.edu.cn;kqzhouysh@hsc.pku.edu.cn
  • Supported by:
    the Beijing Natural Science Foundation(7192228)

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Abstract:

Objective: To investigate the effect of human adipose-derived mesenchymal stem cells (hASCs) exosomes on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) extracted from osteoporotic mice, and to evaluate the effect of hASCs exosomes on preventing bone loss induced by estrogen deficiency. Methods: hASCs exosomes were extracted by ultracentrifugation. The osteoporotic mice were established by bilateral ovariectomy (OVX). BMSCs were isolated from osteo-porotic mice and cultured for further analysis. In the experimental group, these BMSCs were exposed to an osteogenic induction medium supplemented with hASCs exosomes to evaluate their potential effects on osteogenesis. In contrast, the control group was treated with the same osteogenic induction medium, but without the addition of hASCs exosomes, to serve as a baseline comparison for the study. To comprehensively assess the osteogenic differentiation of BMSCs influenced by hASCs exosomes, alkaline phosphatase (ALP) staining, ALP activity quantitative analysis and quantitative reverse transcription polymerase chain reaction (qPCR) were performed. These evaluations provided critical insights into the role of hASCs exosomes in promoting osteoblast differentiation and bone formation in osteoporotic conditions. The fluorescence labeled hASCs exosomes were injected via the tail vein to observe the biodistribution of exosomes. Two weeks after OVX, the mice were divided into three groups: The experimental group consisted of estrogen-deficient mice receiving hASCs exosome injections; the negative control group consisted of estrogen-deficient mice receiving phosphate-buffered saline (PBS) injections; and the positive control group consisted of mice that underwent Sham surgery and received PBS injections.The injections were administered once every 3 days, for a total of 8 injections. Afterward, the femurs were collected from the mice, and micro-computed tomography (micro-CT) was performed to measure bone mineral density and conduct bone morphometric analysis. Results: hASCs exosomes were successfully extracted using ultracentrifugation. After the induction by hASCs exosomes, ALP staining and ALP activity in the BMSCs extracted from osteoporotic mice were significantly enhanced, the expression of osteogenesis related genes in BMSCs were significantly up-regulated. More trabecular bone and higher bone mineral density were observed in estrogen-deficient mice injected with hASCs exosomes compared with estrogen-deficient mice injected with PBS, and there was no significant decrease in bone mineral density compared with the Sham operation group. Conclusion: hASCs exosomes promoted the osteogenic differentiation of BMSCs extracted from osteoporotic mice. hASCs exosomes prevented bone loss induced by estrogen deficiency.

Key words: Mesenchymal stem cells, Osteogenic differentiation, Exosomes, Osteoporosis

CLC Number: 

  • R787

Table 1

Sequences of the primers used for qPCR"

Primers Forward (5′-3′) Reverse (5′-3′)
Gapdh TCACTCAAGATTGTCAGCAA AGATCCACGACGGACACATT
Alp TGACCTTCTCTCCTCCATCC CTTCCTGGGAGTCTCATCCT
Runx2 TAAGAAGAGCCAGGCAGGTG TGGCAGGTACGTGTGGTAGT
Ocn TGCTTGTGACGAGCTATCAG GAGGACAGGGAGGATCAAGT

Figure 1

Characterization of hASCs derived exosomes A, microscopic morphology of hASCs derived exosomes under TEM; B, the size distribution of hASCs derived exosomes measured by NTA; C, Western blot analysis of the exosomal markers, β-tubulin was a negative marker protein. hASCs, human adipose-derived mesenchymal stem cells; TEM, transmission electron microscopy; NTA, nanoparticle tracking analysis."

Figure 2

hASCs exosomes promoted osteogenic differentiation of mBMSCs A, ALP staining on day 3 and day 7; B, expression of osteogenic genes Runx2, Alp, and Ocn in mBMSCs cultured with hASCs exosomes on day 3 and days 7. * *P < 0.01. OM, osteogenic induction medium; EXO, exosomes; hASCs, human adipose-derived mesenchymal stem cells; mBMSCs, mouse bone marrow mesenchymal stem cells; ALP, alkaline phosphatase."

Figure 3

Biodistribution of hASCs exosomes in mice A, the radiant efficiency in hearts, lungs, spleens, livers, kidneys and femurs at 12, 24 and 48 h; B, quantitative analysis of radiant efficiency in femurs at 12, 24 and 48 h; C, the variation of radiant efficiency with time in hearts, lungs, spleens, livers, kidneys and femurs. * *P < 0.01. PBS, phosphate buffered saline; EXO, exosomes; hASCs, human adipose-derived mesenchymal stem cells."

Figure 4

hASCs exosomes prevented bone loss caused by OVX A, micro-CT images and 3D reconstructed images of femurs after treatment of hASCs exosomes; B, HE and Masson staining of mouse femurs after treatment of hASCs exosomes; C, BMD and bone morphometric analyses of mouse femurs after treatment of hASCs exosomes. *P < 0.05, * *P < 0.01. OVX, ovariectomy; PBS, phosphate buffered saline; EXO, exosomes; HE, hematoxylin and eosin; hASCs, human adipose-derived mesenchymal stem cells; BMD, bone mineral density; BV/TV, bone volume/total volume; BS/BV, bone surface area/bone volume; TbN, Trabecular number; TbTh, trabecular thickness; TbSp, trabecular spacing; ns, no significance."

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