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. 2025 Aug 8;26(16):7683.
doi: 10.3390/ijms26167683.

Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Enhance Chondrocyte Function by Reducing Oxidative Stress in Chondrocytes

Che-Wei Wu  1   2   3 , Yao-Hui Huang  1   2 , Pei-Lin Shao  4 , Ling-Hua Chang  1   2 , Cheng-Chang Lu  1   2   5   6   7 , Chung-Hwan Chen  1   2   6   7   8 , Yin-Chih Fu  1   2   6   7 , Mei-Ling Ho  1   2 , Je-Ken Chang  1   2   6   7 , Shun-Cheng Wu  1   2   4
Affiliations

Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Enhance Chondrocyte Function by Reducing Oxidative Stress in Chondrocytes

Che-Wei Wu et al. Int J Mol Sci. .

Abstract

Articular cartilage (AC) has a very limited capacity for self-healing once damaged. Chondrocytes maintain AC homeostasis and are key cells in AC tissue engineering (ACTE). However, chondrocytes lose their function due to oxidative stress. Umbilical cord mesenchymal stem cells (UCMSCs) are investigated as an alternative cell source for ACTE. MSCs are known to regulate tissue regeneration through host cell modulation, largely via extracellular vesicle (EV)-mediated cell-to-cell communication. The purpose of this study was to verify whether UCMSC-derived EVs (UCMSC-EVs) enhance chondrocyte function. The mean particle sizes of the UCMSC-EVs were 79.8 ± 19.05 nm. Transmission electron microscopy (TEM) revealed that UCMSC-EVs exhibited a spherical morphology. The presence of CD9, CD63, and CD81 confirmed the identity of UCMSC-EVs, with α-tubulin undetected. UCMSC-EVs maintained chondrocyte survival, and increased chondrocyte proliferation after intake by chondrocytes. UCMSC-EVs upregulated mRNA levels of SOX-9, collagen type II (Col-II), and Aggrecan, while decreasing collagen type I (Col-I) levels. UCMSC-EVs reduced the oxidative stress of chondrocytes by reducing mitochondrial superoxide production and increasing protein levels of SOD-2 and Sirt-3 in chondrocytes. The 50 most abundant known microRNAs (miRNAs) derived from UCMSC-EVs were selected for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. GO analysis revealed enrichment in pathways associated with small GTPase-mediated signal transduction, GTPase regulatory activity, and mitochondrial matrix. The KEGG analysis indicated that these miRNAs may regulate chondrocyte function through the PI3K-Akt, MAPK, and cAMP signaling pathways. In summary, this study shows that UCMSC-EVs enhance chondrocyte function and may be applied to ACTE.

Keywords: articular cartilage tissue engineering (ACTE); chondrocyte function; extracellular vesicles (EVs); miRNA; oxidative stress; umbilical cord mesenchymal stem cells (UCMSCs).

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of UCMSCs and UCMSC-EVs. (A) Phase-contrast image of UCMSCs cultured on the plastic culture plate. Scale bar: 100 μm. (B) Representative histograms showing the expression of CD105, CD90, CD73, CD45, CD34, and HLA-DR on the surface of UCMSCs. Unstained cells were included as negative controls to validate the specificity of antibody staining. (C) Flow cytometry analysis to quantify the percentage of UCMSC-EVs positive for CD9, CD63, and CD81. (D) Western blot analysis of the protein expressions of CD63, CD81, and α-tubulin in UCMSCs and UCMSC-EVs. (E) Morphology of UCMSC-EVs (arrowhead), as observed using TEM.
Figure 2
Figure 2
Visualization of UCMSC-EVs internalization in chondrocytes. Chondrocytes were treated with UCMSC-EVs at a concentration of 0 (Control group) or 1 × 109 particles/mL (UCMSC-EVs group) for 7 days. Uptake of UCMSC-EVs by chondrocytes was subsequently evaluated. Uptake of red-fluorescence-labeled UCMSC-EVs in chondrocytes was detected by fluorescence microscopy, and images were obtained using a camera. Green fluorescence stain, cytoplasm; blue fluorescence stain, cell nucleus; red fluorescence stain, ExoSparker-stained UCMSC-EVs. Scale bar: 100 μm.
Figure 3
Figure 3
Effect of UCMSC-EVs on survival and proliferation of chondrocytes. Chondrocytes were treated with UCMSC-EVs at concentrations of 0 (Control group) or 108–1010 particles/mL (UCMSC-EVs group) for 7 days. (A) Live/dead cell staining was performed on day 7 to assess cell viability. Live cells were labeled with calcein-AM (green), while dead cells were stained with ethidium homodimer-1 (red). Green fluorescence indicated viable cells, and red fluorescence indicated non-viable cells. Chondrocytes remained viable following UCMSC-EVs treatment. (B) Quantitative analysis of cell survival revealed no detectable cell death after UCMSC-EVs exposure. (C) Chondrocyte proliferation was evaluated using an MTS assay on day 7. UCMSC-EVs significantly promoted chondrocyte proliferation, especially at concentrations of 109 and 1010 particles/mL. Data are expressed as mean ± SEM (n = 6). * p < 0.05, ** p < 0.01 vs. Control group.
Figure 4
Figure 4
Effect of UCMSC-EVs on mRNA expression of chondrogenic genes (SOX-9, Col-II, and Aggrecan) and fibrocartilaginous gene (Col-I) of chondrocytes. Chondrocytes were cultured for 7 days with UCMSC-EVs at concentrations of 0 (Control group) or 1 × 109 to 1 × 1010 particles/mL (UCMSC-EVs group). On day 7, mRNA expression levels of chondrogenic markers ((A) SOX-9, (B) Col-II, (C) Aggrecan) and the fibrocartilaginous marker ((D) Col-I) were analyzed. Total RNA was isolated and subjected to real-time polymerase chain reaction analysis. Gene expression levels are presented relative to the Control group, which is normalized to 1. Data are expressed as mean ± standard error of the mean (SEM; n = 4). * p < 0.05 and ** p < 0.01 for comparisons with the Control group.
Figure 5
Figure 5
Effect of UCMSC-EVs on oxidative stress of chondrocytes. Chondrocytes were treated with UCMSC-EVs at concentrations of 0 (Control group) or 108–1010 particles/mL (UCMSC-EVs group) for 7 days to assess oxidative stress. (A) Mitochondrial superoxide levels were evaluated by quantifying MitoSOX Red fluorescence intensity (n = 6). (B,C) Protein expression levels of SOD-2 and Sirt-3 were analyzed by Western blotting following treatment with 1010 particles/mL UCMSC-EVs. β-actin was used as the internal control. Protein expression data are normalized to the Control group (defined as 1) and presented as mean ± standard error of the mean (SEM; n = 3–5). * and ** indicate p < 0.05 and p < 0.01, respectively, compared to the chondrocytes in the Control group.
Figure 6
Figure 6
miRNA bioinformatics analysis of UCMSC-EVs. Small RNA sequencing was performed on three independent batches of UCMSC-EVs. The 50 most abundant known miRNAs identified in the UCMSC-EVs were ranked based on total read counts across all replicates (n = 3), as shown in the figure.
Figure 7
Figure 7
GO enrichment pathway analysis. GO pathway analysis was performed for target genes of miRNAs enriched in UCMSC-EVs. GO biological process (BP), cellular component (CC), and molecular function (MF) terms are presented. Bar and dot plots of GO are shown (n = 3).
Figure 7
Figure 7
GO enrichment pathway analysis. GO pathway analysis was performed for target genes of miRNAs enriched in UCMSC-EVs. GO biological process (BP), cellular component (CC), and molecular function (MF) terms are presented. Bar and dot plots of GO are shown (n = 3).
Figure 7
Figure 7
GO enrichment pathway analysis. GO pathway analysis was performed for target genes of miRNAs enriched in UCMSC-EVs. GO biological process (BP), cellular component (CC), and molecular function (MF) terms are presented. Bar and dot plots of GO are shown (n = 3).
Figure 8
Figure 8
KEGG pathway analysis. KEGG pathway analyses were conducted for predicted target genes of miRNAs enriched in UCMSC-EVs. Representative bar and dot plots summarizing the enriched pathways are presented (n = 3).
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