Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan-Dec:30:963689720986142.
doi: 10.1177/0963689720986142.

Low-Intensity Pulsed Ultrasound Promotes Autophagy-Mediated Migration of Mesenchymal Stem Cells and Cartilage Repair

Peng Xia  1 , Xinwei Wang  1 , Qi Wang  1 , Xiaoju Wang  1 , Qiang Lin  1 , Kai Cheng  1 , Xueping Li  1
Affiliations

Low-Intensity Pulsed Ultrasound Promotes Autophagy-Mediated Migration of Mesenchymal Stem Cells and Cartilage Repair

Peng Xia et al. Cell Transplant. 2021 Jan-Dec.

Abstract

Mesenchymal stem cell (MSC) migration is promoted by low-intensity pulsed ultrasound (LIPUS), but its mechanism is unclear. Since autophagy is known to regulate cell migration, our study aimed to investigate if LIPUS promotes the migration of MSCs via autophagy regulation. We also aimed to investigate the effects of intra-articular injection of MSCs following LIPUS stimulation on osteoarthritis (OA) cartilage. For the in vitro study, rat bone marrow-derived MSCs were treated with an autophagy inhibitor or agonist, and then they were stimulated by LIPUS. Migration of MSCs was detected by transwell migration assays, and stromal cell-derived factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR4) protein levels were quantified. For the in vivo study, a rat knee OA model was generated and treated with LIPUS after an intra-articular injection of MSCs with autophagy inhibitor added. The cartilage repair was assessed by histopathological analysis and extracellular matrix protein expression. The in vitro results suggest that LIPUS increased the expression of SDF-1 and CXCR4, and it promoted MSC migration. These effects were inhibited and enhanced by autophagy inhibitor and agonist, respectively. The in vivo results demonstrate that LIPUS significantly enhanced the cartilage repair effects of MSCs on OA, but these effects were blocked by autophagy inhibitor. Our results suggest that the migration of MSCs was enhanced by LIPUS through the activation autophagy, and LIPUS improved the protective effect of MSCs on OA cartilage via autophagy regulation.

Keywords: autophagy; mesenchymal stem cell; migration; ultrasound.

PubMed Disclaimer

Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Effects of 3-MA and rapamycin on MSC autophagy. (A) Electron microscopy depicting autophagosomes (arrows); scale bars = 0.5 μm. (B) Immunofluorescence staining depicting LC3+ cells (green); scale bars = 50 μm. (C) Western blot analysis of Beclin1, LC3I, LC3II, and β-actin expression in MSCs. The values are the mean ± SD; n = 4, *P < 0.05. 3-MA: 3-methyladenine; MSC: mesenchymal stem cell.
Figure 2.
Figure 2.
Effects of 3-MA and rapamycin on MSC migration. (A) Western blot analysis of SDF-1, CXCR4, and β-actin expression in MSCs. (B) The transwell migration capacity of MSCs stained with crystal violet, and the bar graph comparing the counts of migrated cells; scale bars = 50μm. The values are the mean ± SD; n = 4, *P < 0.05. 3-MA: 3-methyladenine; CXCR4: C-X-C chemokine receptor type 4; MSC: mesenchymal stem cell; SDF-1: stromal cell-derived factor-1.
Figure 3.
Figure 3.
Effects of LIPUS on autophagy in MSCs. (A) Electron microscopy depicting autophagosomes (arrows); scale bars = 0.5 μm. (B) Immunofluorescence staining depicting LC3+ cells (green); scale bars = 50 μm. (C) Western blot analysis of Beclin1, LC3I, LC3II, and β-actin expression in MSCs stimulated with 4 intensities of LIPUS. The values are the mean ± SD; n = 4, *P < 0.05. 3-MA: 3-methyladenine; LIPUS: low-intensity pulsed ultrasound; MSC: mesenchymal stem cell.
Figure 4.
Figure 4.
Effects of LIPUS on the migration of MSCs. (A) Western blot analysis of SDF-1, CXCR4, and β-actin expression in MSCs. (B) The transwell migration capacity of MSCs stained with crystal violet and the bar graph comparing the counts of migrated cells; scale bars = 50 μm. The values are the mean ± SD; n = 4, *P < 0.05. CXCR4: C-X-C chemokine receptor type 4; LIPUS: low-intensity pulsed ultrasound; MSC: mesenchymal stem cell; SDF-1: stromal cell-derived factor-1.
Figure 5.
Figure 5.
Effects of LIPUS on autophagy in MSCs treated with 3-MA or rapamycin. (A) Electron microscopy depicting autophagosomes (arrows); scale bars = 0.5 μm. (B) Immunofluorescence staining depicting LC3+ cells (green); scale bars = 50 μm. (C) Western blot analysis of Beclin1, LC3I, LC3II, and β-actin expression in MSCs stimulated with 4 intensities of LIPUS. Values are the mean ± SD; n = 4, *P < 0.05. 3-MA: 3-methyladenine; LIPUS: low-intensity pulsed ultrasound; MSC: mesenchymal stem cell.
Figure 6.
Figure 6.
Effects of LIPUS on the migration of MSCs treated with 3-MA or rapamycin. (A) Western blot analysis of SDF-1, CXCR4, and β-actin expression in MSCs. (B) The transwell migration capacity of MSCs stained with crystal violet and the bar graph comparing the counts of migrated cells; scale bars = 50 μm. The values are the mean ± SD; n = 4, *P < 0.05. 3-MA, 3-methyladenine; CXCR4: C-X-C chemokine receptor type 4; LIPUS: low-intensity pulsed ultrasound; MSC: mesenchymal stem cell; SDF-1: stromal cell-derived factor-1.
Figure 7.
Figure 7.
Effects of LIPUS stimulation after intra-articular injection of MSCs on OA cartilage. (A) Morphological changes of cartilage revealed by safranin-O/fast green staining and observed under a microscope; scale bars = 1000 μm, 200 μm. (B) Bar graph comparing the Mankin scores. (C) Bar graph comparing the TAC thickness (μm). (D) Western blot analysis of COL2, AGG, and β-actin expression in cartilage. The values are the mean ± SD; n = 4, *P < 0.05. LIPUS: low-intensity pulsed ultrasound; MSC: mesenchymal stem cell; OA: osteoarthritis; TAC: total articular cartilage.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wang Y, Yuan M, Guo Q, Lu S, Peng J. Mesenchymal stem cells for treating articular cartilage defects and osteoarthritis. Cell Transplant. 2015;24(9):1661–1678. - PubMed
    1. Short B, Brouard N, Occhiodoro-Scott T, Ramakrishnan A, Simmons PJ. Mesenchymal stem cells. Arch Med Res. 2003;34(6):565–571. - PubMed
    1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284(5411):143–147. - PubMed
    1. Quintavalla J, Uziel-Fusi S, Yin J, Boehnlein E, Pastor G, Blancuzzi V, Singh HN, Kraus KH, O’Byrne E, Pellas TC. Fluorescently labeled mesenchymal stem cells (mscs) maintain multilineage potential and can be detected following implantation into articular cartilage defects. Biomaterials. 2002;23(1):109–119. - PubMed
    1. Pittenger MF, Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res. 2004;95(1):9–20. - PubMed

Publication types