Low-intensity pulsed ultrasound promotes mesenchymal stem cell transplantation-based articular cartilage regeneration via inhibiting the TNF signaling pathway

Abstract

Background: Mesenchymal stem cell (MSC) transplantation therapy is highly investigated for the regenerative repair of cartilage defects. Low-intensity pulsed ultrasound (LIPUS) has the potential to promote chondrogenic differentiation of MSCs. However, its underlying mechanism remains unclear. Here, we investigated the promoting effects and mechanisms underlying LIPUS stimulation on the chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) and further evaluated its regenerative application value in articular cartilage defects in rats.

Methods: LIPUS was applied to stimulate cultured hUC-MSCs and C28/I2 cells in vitro. Immunofluorescence staining, qPCR analysis, and transcriptome sequencing were used to detect mature cartilage-related markers of gene and protein expression for a comprehensive evaluation of differentiation. Injured articular cartilage rat models were established for further hUC-MSC transplantation and LIPUS stimulation in vivo. Histopathology and H&E staining were used to evaluate the repair effects of the injured articular cartilage with LIPUS stimulation.

Results: The results showed that LIPUS stimulation with specific parameters effectively promoted the expression of mature cartilage-related genes and proteins, inhibited TNF-α gene expression in hUC-MSCs, and exhibited anti-inflammation in C28/I2 cells. In addition, the articular cartilage defects of rats were significantly repaired after hUC-MSC transplantation and LIPUS stimulation.

Conclusions: Taken together, LIPUS stimulation could realize articular cartilage regeneration based on hUC-MSC transplantation due to the inhibition of the TNF signaling pathway, which is of clinical value for the relief of osteoarthritis.

Keywords: Articular cartilage regeneration; Chondrogenic differentiation; Low-intensity pulsed ultrasound (LIPUS); Mesenchymal stem cells (MSCs); TNF signaling pathway.

Fig. 1

Identification of hUC-MSCs and the effect of LIPUS on the proliferation of hUC-MSCs. a The six-channel ultrasound stimulation system used for in vitro LIPUS stimulation. b LIPUS parameters. c The tri-lineage differentiation capability identification of hUC-MSCs; scale bar: 200 μm. d Flow cytometric identification of specific surface markers of hUC-MSCs. e Flow cytometry analysis for cell apoptosis of hUC-MSCs stimulated with LIPUS. f Flow cytometry analysis for cell cycle distribution of hUC-MSCs stimulated with LIPUS. g Representative live-dead staining images of hUC-MSCs stimulated with LIPUS; scale bar: 200 μm. h Quantification data for apoptosis rate of hUC-MSCs stimulated with LIPUS. i Quantification data for the cell cycle of hUC-MSCs stimulated with LIPUS

Fig. 2

Effects of LIPUS on the chondrogenic differentiation of hUC-MSCs. The hUC-MSCs were stimulated by 20 min/day LIPUS stimulation for 7 days before analyses. a Representative Alcian blue staining images showing the differentiation level of hUC-MSCs into chondrocytes; scale bar: 200 μm. b Quantification data of the proteoglycan production for the Alcian blue staining results. c–e Representative immunofluorescence images showing the expression of cytoskeleton actin (c), and two marker genes of chondrogenic differentiation, SOX-9 (d) and ACAN (e); scale bar: 100 μm. f qPCR analysis of the expression of SOX-9, COL-II, COL-X, ACAN, and COMP. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3)

Fig. 3

Effects of LIPUS stimulation on the proliferation and inflammation of C28/I2 cells. a Flow cytometry analysis for cell apoptosis of C28/I2 cells stimulated with LIPUS. b Quantification data for apoptosis rate of C28/I2 cells. c Flow cytometry analysis for cell cycle distribution of C28/I2 cells. d Quantification data for the cell cycle of C28/I2 cells. e Representative live-dead staining images of C28/I2 stimulated with LIPUS; scale bar: 200 μm. f qPCR analysis of the expression of TNF-α, IL-1β, and IL-6 in C28/I2 cells, after LPS treatment (to produce inflammation) and LIPUS stimulation. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3)

Fig. 4

Transcriptome sequencing analysis. a Cluster heat map of mRNA expression of Ctrl, US-70, and US-100 samples. b A volcano plot comparing the DEGs between the US-70 and Ctrl groups (Down-regulated genes showed in green and up-regulated genes showed in red; cutoff: p-value < 0.05 and |log₂ FC|> 1). c–e KEGG pathway term analysis (c), KEGG pathway analysis (d), and GO pathway analysis of the DEGs (e) between the US-70 and Ctrl groups. f PPI network analysis of the core regulatory genes of the DEGs between the US-70 and Ctrl groups

Fig. 5

Effects of TNF signaling pathway in LIPUS stimulation-induced hUC-MSC chondrogenic differentiation. a qPCR analysis of the expression of inflammatory factor genes TNF-α, IL-1β, and CXCL8. *p < 0.05, **p < 0.01, ***p < 0.001 (n = 3). b Representative immunofluorescence images showing the expression level of inflammatory factor TNF-α; scale bars: 50 μm and 100 μm, respectively. c Quantification data of the proteoglycan production for the Alcian blue staining results. d Representative Alcian blue staining images showing the differentiation level of hUC-MSCs into chondrocytes; scale bar: 200 μm. e–f Representative immunofluorescence images showing the expression of the chondrogenic differentiation markers SOX-9 (e) and COL-II (f); scale bar: 100 μm

Fig. 6

Validation of LIPUS and hUC-MSC-based cartilage regeneration in vivo. a Top—the ultrasound stimulation system for in vivo LIPUS stimulation on the rat knee joint. Bottom—partially enlarged photograph of the experiment setup. b Bright-field photography showing the appearance of rat knee joint samples after LIPUS stimulation for 2, 4, and 6 weeks; scale bar: 1 mm. In each group, left—overall view of the knee joint, right—partially enlarged photograph of the defect area. c Respective H&E staining and Safranin O-fast green staining of the paraffin sections of the knee joint defect showing the healing effect of LIPUS stimulation; scale bars: 500 μm for H&E staining and 200 μm for Safranin O-fast green staining, respectively. d Representative immunohistochemistry images of COL-II, TNF-α, and CD44 of the knee joint defect in rats after 6 weeks of LIPUS stimulation; scale bar: 500 μm. e Representative H&E staining images of major organs in rats after 6 weeks of LIPUS stimulation; scale bar: 500 μm

Fig. 7

Schematic representation of LIPUS stimulation and hUC-MSC transplantation-based articular cartilage regeneration. Created with BioRender.com