TGF-β1-induced chondrogenesis of bone marrow mesenchymal stem cells is promoted by low-intensity pulsed ultrasound through the integrin-mTOR signaling pathway

Abstract

Background: Low-intensity pulsed ultrasound (LIPUS) is a mechanical stimulus that plays a key role in regulating the differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the way in which it affects the chondrogenic differentiation of BMSCs remains unknown. In this study, we aimed to investigate whether LIPUS is able to influence TGF-β1-induced chondrogenesis of BMSCs through the integrin-mechanistic target of the Rapamycin (mTOR) signaling pathway.

Methods: BMSCs were isolated from rat bone marrow and cultured in either standard or TGF-β1-treated culture medium. BMSCs were then subjected to LIPUS at a frequency of 3 MHz and a duty cycle of 20%, and integrin and mTOR inhibitors added in order to analyze their influence on cell differentiation. BMSCs were phenotypically analyzed by flow cytometry and the degree of chondrogenesis evaluated through toluidine blue staining, immunofluorescence, and immunocytochemistry. Furthermore, expression of COL2, aggrecan, SOX9, and COL1 was assessed by qRT-PCR and western blot analysis.

Results: We found that LIPUS promoted TGF-β1-induced chondrogenesis of BMSCs, represented by increased expression of COL2, aggrecan and SOX9 genes, and decreased expression of COL1. Notably, these effects were prevented following addition of integrin and mTOR inhibitors.

Conclusions: Taken together, these results indicate that mechanical stimulation combined with LIPUS promotes TGF-β1-induced chondrogenesis of BMSCs through the integrin-mTOR signaling pathway.

Keywords: Bone marrow mesenchymal stem cells (BMSCs); Differentiation; Integrin; Low-intensity pulsed ultrasound (LIPUS); mTOR.

Fig. 1

LIPUS stimulation of BMSCs in vitro. A layer of coupling agent (< 1 mm thick) was applied between the LIPUS probe and culture dish, the probe placed below the tissue culture dish. BMSCs were treated once a day for 10 days at a duty cycle of 20%, at 3 MHz for 20 minutes in a 37 °C incubator in a humidified atmosphere with 5% CO₂ in air. LIPUS low-intensity pulsed ultrasound. BMSCs bone marrow mesenchymal stem cells, LIPUS low-intensity pulsed ultrasound

Fig. 2

General observation and identification of BMSCs and TGF-β1-induced chondrogenesis. a Second generation of BMSCs cultured in basic medium and observation of cells in vitro by microscopy on days 5 and 10; scale bars = 50 μm. b Flow cytometry histogram of BMSCs. Purple shaded area represents control, red curve represents cells positive for CD markers. Graphs a, b, c, d separately indicate markers of CD34 (0.21 ± 0.03%), CD45 (0.63 ± 0.09%), CD90 (72.00 ± 0.67%), and CD44 (66.86 ± 0.36%), respectively

Fig. 3

Identification of TGF-β1-induced chondrogenesis. a Immunofluorescence staining of COL2 and toluidine blue staining in BMSCs cultured with basic or chondrogenic medium (basic medium treated with different doses of TGF-β1) on day 10; scale bars = 100 μm or 25 μm. b qRT-PCR analysis of COL2, aggrecan, SOX9, and COL1. The values are mean ± SEM of triplicate experiments. n = 4; *P < 0.05. COL1 collagen type-I, COL2 collagen type-2. GAPDH glyceraldehyde 3-phosphate dehydrogenase, LIPUS low-intensity pulsed ultrasound, SOX9 SRY-related high mobility group-box gene 9, TGF transforming growth factor

Fig. 4

LIPUS promoted TGF-β1-induced chondrogenesis of BMSCs. a Immunocytochemistry staining of COL2 in BMSCs cultured with 10 ng/ml TGF-β1-treated medium after LIPUS stimulation at four intensities on days 5 and 10. Images were acquired using a microscope; scale bars = 25 μm. b Western blot analysis of COL2, aggrecan, SOX9, and COL1 with β-actin as a loading control. c Statistics of the western blot results of COL2, aggrecan, SOX9, and COL1. The ratio between the target and β-actin was used to normalize the data for comparison. Values are the mean ± SEM of triplicate experiments. n = 4; *P < 0.05. COL1 collagen type-I, COL2 collagen type-2. LIPUS low-intensity pulsed ultrasound, SOX9 SRY-related high mobility group-box gene 9, TGF transforming growth factor

Fig. 5

Inhibition of integrin-mTOR pathway suppressed TGF-β1-induced chondrogenesis of BMSCs and the treatment effects of LIPUS. a Western blot analysis demonstrate that GRGDSP inhibited β1 integrin expression in a dose-dependent manner. b Western blot results show that Rapamycin inhibited the activation of mTOR in a dose-dependent manner. c Immunohistochemical staining for COL2 of BMSCs cultured with TGF-β1-treated medium in control, LIPUS, GRGDSP, GRGDSP + LIPUS, Rapamycin, and Rapamycin + LIPUS groups. Images acquired by microscopy on day 10; scale bars = 25 μm. d Western blot analysis of COL2, aggrecan, SOX9, COL1, integrin β1, and p-mTOR with β-actin as a loading control in BMSCs treated with TGF-β1 with or without LIPUS stimulation following GRGDSP or Rapamycin treatment. e–j Statistics of the western blot results of COL2, aggrecan, SOX9, COL1, β1 integrin, and p-mTOR. The ratio between the target and β-actin was used to normalize the data for comparison. The values are mean ± SEM of triplicate experiments. n = 6; *P < 0.05. COL1 collagen type-I, COL2 collagen type-2. LIPUS low-intensity pulsed ultrasound, mTOR mechanistic target of rapamycin, SOX9 SRY-related high mobility group-box gene 9, TGF transforming growth factor