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. 2019 Jan 22;10(1):41.
doi: 10.1186/s13287-019-1142-z.

Low-intensity pulsed ultrasound promotes chondrogenesis of mesenchymal stem cells via regulation of autophagy

Xiaoju Wang  1 , Qiang Lin  1 , Tingting Zhang  1 , Xinwei Wang  1 , Kai Cheng  1 , Mingxia Gao  1 , Peng Xia  2 , Xueping Li  3
Affiliations

Low-intensity pulsed ultrasound promotes chondrogenesis of mesenchymal stem cells via regulation of autophagy

Xiaoju Wang et al. Stem Cell Res Ther. .

Abstract

Background: Low-intensity pulsed ultrasound (LIPUS) can induce mesenchymal stem cell (MSC) differentiation, although the mechanism of its potential effects on chondrogenic differentiation is unknown. Since autophagy is known to regulate the differentiation of MSCs, the aim of our study was to determine whether LIPUS induced chondrogenesis via autophagy regulation.

Methods: MSCs were isolated from the rat bone marrow, cultured in either standard or chondrogenic medium, and stimulated with 3 MHz of LIPUS given in 20% on-off cycles, with or without prior addition of an autophagy inhibitor or agonist. Chondrogenesis was evaluated on the basis of aggrecan (AGG) organization and the amount of type II collagen (COL2) and the mRNA expression of AGG, COL2, and SRY-related high mobility group-box gene 9 (SOX9) genes.

Results: LIPUS promoted the chondrogenic differentiation of MSCs, as shown by the changes in the extracellular matrix (ECM) proteins and upregulation of chondrogenic genes, and these effects were respectively augmented and inhibited by the autophagy inhibitor and agonist.

Conclusions: Taken together, these results indicate that LIPUS promotes MSC chondrogenesis by inhibiting autophagy.

Keywords: Autophagy; Chondrogenesis; Low-intensity pulsed ultrasound (LIPUS); Mesenchymal stem cells (MSCs).

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Conflict of interest statement

Authors’ information

All the authors are researchers at Department of Rehabilitation Medicine, Nanjing First Hospital, Nanjing Medical University, China. Peng Xia and Xueping Li are the leaders of our research group.

Ethics approval and consent to participate

The experimental protocol relating to rats was approved by the Nanjing Medical University Ethics Committee of Nanjing Hospital (20150829).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Characterization of MSCs and the effects of autophagy agonist and inhibitor. The MSCs were cultured in basic medium for 10 days before analyses. a Representative image showing second generation MSCs on day 10 of culture; scale bars = 50 μm. Flow cytometry histograms showing percentage of MSC marker-positive cells (red curve); purple shaded area represents the control. b Immunoblots showing the levels of Beclin1, LC3I, and LC3II in MSCs, with β-actin as the loading control. c Representative electron microscopy images showing autophagosomes (arrows); scale bars = 0.5 μm. d Left panel—representative immunofluorescence images showing LC3+ cells (green); scale bars = 50 μm. Right panel—bar graph comparing the number of LC3+ cells. The values are the mean ± SD of triplicate experiments; *P < 0.05
Fig. 2
Fig. 2
Effects of LIPUS on autophagy in MSCs. The MSCs were cultured in basic medium or chondrogenic medium for 10 days before analyses. a Immunoblots showing the levels of Beclin1, LC3I, and LC3II in differentiating MSCs stimulated with varying intensities of LIPUS, with β-actin as the loading control. b Representative electron microscopy images showing autophagosomes (arrows); scale bars = 0.5 μm. c Left panel—representative immunofluorescence images showing LC3+ cells (green); scale bars = 50 μm. Right panel—bar graph comparing the number of LC3+ cells. The values are the mean ± SD of triplicate experiments; *P < 0.05
Fig. 3
Fig. 3
Effects of LIPUS on the chondrogenesis of MSCs. The MSCs were cultured in basic medium or chondrogenic medium for 10 days before analyses. a Representative images of ICC showing COL2+ cells (upper panel), and toluidine blue-staining showing AGG (lower panel) in differentiating MSCs stimulated with varying intensities of LIPUS; scale bars = 100 μm. b–d Bar graphs showing relative levels of COL2 (b), AGG (c), and SOX9 (d) mRNA in LIPUS-stimulated and unstimulated MSCs. The values are the mean ± SD of triplicate experiments; *P < 0.05
Fig. 4
Fig. 4
Effects of LIPUS on autophagy in MSCs treated with autophagy agonist or inhibitor. The MSCs were cultured in chondrogenic medium for 10 days before analyses. a Immunoblots showing the levels of Beclin1, LC3I, and LC3II in differentiating MSCs stimulated with varying intensities of LIPUS, with β-actin as the loading control. b Representative electron microscopy images showing autophagosomes (arrows); scale bars = 0.5 μm. c Left panel—representative immunofluorescence images showing LC3+ cells (green); scale bars = 50 μm. Right panel—bar graph comparing the number of LC3+ cells. The values are the mean ± SD of triplicate experiments; *P < 0.05
Fig. 5
Fig. 5
Effects of LIPUS on the chondrogenesis of MSCs treated with autophagy agonist or inhibitor. a Representative images of immunocytochemistry staining of COL2 (upper panel) and toluidine blue staining (lower panel) in differentiated MSCs stimulated with varying intensities of LIPUS; scale bars = 100 μm. b–d Bar graphs showing relative levels of COL2 (b), AGG (c), and SOX9 (d) mRNA in LIPUS-stimulated and unstimulated MSCs. The values are the mean ± SD of triplicate experiments; *P < 0.05
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