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. 2024 Apr;28(8):e18327.
doi: 10.1111/jcmm.18327.

Cartilage stem/progenitor cells-derived exosomes facilitate knee cartilage repair in a subacute osteoarthritis rat model

Jing Chen  1   2 , Xiaohui Ni  3 , Jian Yang  2 , Hongwei Yang  4 , Xiaoyu Liu  2 , Minhao Chen  1 , Cheng Sun  2 , Youhua Wang  1
Affiliations

Cartilage stem/progenitor cells-derived exosomes facilitate knee cartilage repair in a subacute osteoarthritis rat model

Jing Chen et al. J Cell Mol Med. 2024 Apr.

Abstract

Cartilage defects in the knee are often associated with the progression of degenerative osteoarthritis (OA), and cartilage repair is a useful strategy for managing this disease. However, cartilage repair is challenging because of the unique environment within the tissue. Recently, stem cell-based therapies have shed new light on this issue. In this study, we prepared exosomes (EXOs) from cartilage stem/progenitor cells (CSPCs) and found that treatment with EXOs increased the viability, migration, and proliferation of cultured primary chondrocytes. In a subacute OA rat model, the application of EXOs facilitated cartilage regeneration as evidenced by histological staining. Exosomal protein analysis together with bioinformatics suggested that cyclin-dependent kinase 9 (CDK9) is a key factor for chondrocyte growth and migration. Functional studies confirmed this prediction, that is, inhibiting CDK9 reduced the beneficial effects induced by EXOs in primary chondrocytes; while overexpression of CDK9 recapitulated the EXOs-induced phenotypes. RNA-Seq data showed that a set of genes involved in cell growth and migration were up-regulated by EXOs in chondrocytes. These changes could be partially reproduced by CDK9 overexpression. Overall, our data suggest that EXOs derived from primary CSPCs hold great therapeutic potential for treating cartilage defect-associated disorders such as degenerative OA, and that CDK9 is a key factor in this process.

Keywords: CDK9; cartilage repair; cartilage stem/progenitor cells; chondrocytes; exosomes.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

FIGURE 1
FIGURE 1
Preparation and characterization of cartilage stem/progenitor cells (CSPCs)‐derived EXOs. (A) Representative transmission electron microscopy (TEM) images of CSPC‐EXOs (scale bar = 200 nm). (B) Nanoparticle tracking analysis of CSPC‐EXOs. (C) Western blot analysis showing exosomal markers CD9 and CD63. Calnexin was used as a negative control. (D) Identification of rat primary chondrocytes. Cell morphology was observed with a light microscope (scale bar = 100 μm). Immunofluorescence analysis showing COL II (green) expression. DAPI (blue) was used to label the cell nuclei. Scale bar = 50 μm. (E) Internalization of CSPC‐EXOs in rat primary chondrocytes. CSPC‐EXOs were labelled with PKH26 (red) and co‐cultured with primary chondrocytes. FITC‐Phalloidin (green) and DAPI (blue) were used to label the cytoskeleton and the cell nuclei, respectively. Scale bar = 100 μm or 25 μm as indicated.
FIGURE 2
FIGURE 2
CSPC‐EXOs improve cell viability, migration, and proliferation in chondrocytes. (A) CSPC‐EXOs stimulate cell viability in primary chondrocytes. Cells were treated with EXOs at different dosages as indicated for 24 h, and then cell viability was analysed with a CCK‐8 kit. n = 5. (B) CSPC‐EXOs promote cell migration. Primary chondrocytes were treated with CSPC‐EXOs (1 × 109 p/mL) for 12 h or 24 h, cell migration was analysed by wound healing assay. Scale bar = 200 μm. (C–D) CSPC‐EXOs counteract the decreased cell viability induced by IL‐1β in chondrocytes. Primary chondrocytes were treated with EXOs (1 × 109 p/mL) and IL‐1β (10 ng/mL) as indicated for 12 h (C) and 24 h (D), cells were then subjected to cell viability assay with a CCK‐8 kit. n = 4–6. (E) CSPC‐EXOs improve cell proliferation in chondrocytes treated with IL‐1β. Primary chondrocytes were pretreated with CSPC‐EXOs at the concentrations of 1 × 109 p/mL. 6 h post‐pretreatment, cells were incubated with 10 ng/mL of IL‐1β for 24 h and 48 h, EdU incorporation was used to analyse cell proliferation. Scale bar = 400 μm. (F) Quantification of cell proliferation as shown in (E). n = 3. Values are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one‐way ANOVA.
FIGURE 3
FIGURE 3
CSPC‐EXOs improve cartilage repair in subacute OA model rats. (A) Experimental timeline. (B) Macroscopic appearance of rat knee joints. MIA, monosodium iodoacetate.
FIGURE 4
FIGURE 4
Representative images of haematoxylin and eosin (A), Safranin O/Fast Green (B) and Toluidine blue (C) staining of knee joint specimens. Scale bar = 500 μm (40×) or 200 μm (100×).
FIGURE 5
FIGURE 5
Immunohistochemical staining of Aggrecan (A), COL‐I (B) and COL‐II (C) in cartilage tissues. Scale bar = 500 μm (40×) or 200 μm (100×).
FIGURE 6
FIGURE 6
Exosomal protein identification. (A) The work flow of proteomic analysis. (B) The Eukaryotic Orthologous Group (KOG) functional classification. (C) The peptide fragments, m/z values, and retention time (RT) of CDK9 in mass spectrometry.
FIGURE 7
FIGURE 7
Inhibition of CDK9 diminishes CSPC‐EXOs induced cell viability, migration, and proliferation. (A–B) Inhibition of CDK9 by NVP‐2 mitigates the increased cell viability induced by CSPC‐EXOs. Primary chondrocytes were pretreated with NVP‐2 (2 μM) for 3 h, and then CSPC‐EXOs was added as indicated. After 24 h (A), and 48 h (B), cell viability was assayed with a CCK‐8 kit. n = 6. (C) NVP‐2 diminishes the enhanced cell proliferation induced by CSPC‐EXOs. Cell treatments were described in (A–B). Cell proliferation was analysed by EdU staining. Scale bar = 400 μm. (D) Quantification of cell proliferation as shown in (C). n = 3. (E) NVP‐2 counteracts CSPC‐EXOs induced cell migration. Cell treatments were described in (A–B). Cell migration was analysed by wound healing assay. (F) Quantification of healing area as shown in (E). n = 3. Values are presented as mean ± SEM. *p < 0.05, and ***p < 0.001, one‐way ANOVA.
FIGURE 8
FIGURE 8
Overexpression of CDK9 prevents the reduced cell viability, migration, and proliferation in chondrocytes treated with IL‐1β. (A) Overexpression of CDK9 in chondrocytes. Primary chondrocytes were transfected with a plasmid expressing CDK9 by electroporation. 24 h post‐transfection, cells were harvested for western blot analysis. Actin was used as a loading control. EV, empty vector. (B–C) CDK9 improves cell viability. Primary chondrocytes were transfected with a plasmid expressing CDK9, 6 h post‐transfection, cells were treated with IL‐1β (10 ng/mL) for additional 24 h (B) and 48 h (C). Cell viability was examined with a CCK‐8 kit. n = 5. (D) CDK9 stimulates cell proliferation. Cell treatments were described in (B–C). Cell proliferation was assayed by EdU incorporation. Scale bar = 400 μm. (E) Quantification of cell proliferation as shown in (D). n = 3. (F) CDK9 induces cell migration. Cell treatments were described in (B–C). Cell migration was tested by wound healing assay. Scale bar = 200 μm. (G) Quantification of cell migration as shown in (F). n = 3. Values are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one‐way ANOVA.
FIGURE 9
FIGURE 9
EXOs derived from CDK9‐overexpressed CSPCs have better performances in chondrocyte viability and migration. (A) Exosomal CDK9 was increased in EXOs derived from CDK9‐overexpressed CSPCs. EXOs were prepared from CSPCs transfected with a plasmid expressing Cdk9 (CDK9‐EXOs) or empty vector (EV‐EXOs). CDK9 expression was analysed by western blot, and CD63 was used as a loading control. EV, empty vector. (B, C) CDK9‐EXOs increase the viability of chondrocytes. Primary chondrocytes were treated with IL‐1β (10 ng/mL), EV‐EXOs, and CDK9‐EXOs as indicated for 24 h (B) and 48 h (C). Cell viability was examined with a CCK‐8 kit. n = 6. (D) CDK9‐EXOs stimulates the migration of chondrocytes. Cell treatments were described in (B, C). Cell migration was tested by wound healing assay. Scale bar = 200 μm. (E) Quantification of cell migration as shown in (D). n = 3. Values are presented as mean ± SEM. *p < 0.05, ***p < 0.001, one‐way ANOVA.
FIGURE 10
FIGURE 10
RNA‐Seq analysis showing differentially expressed genes induced by CSPC‐EXOs. Primary chondrocytes were treated with CSPC‐EXOs for 24 and 48 h, cells were harvested for RNA‐Seq. (A, B) The up‐regulated genes with potential role in cell proliferation (A) and cell migration (B).
FIGURE 11
FIGURE 11
Effects of CDK9 on gene expression involved in cell proliferation and migration. Primary chondrocytes were transfected with the plasmid expressing CDK9 by electroporation. 24 and 48 h post‐transfection, cells were harvested for qPT‐PCR analysis, 18S rRNA was used as a house‐keeping gene. Values are presented as mean ± SEM. n = 3. *p < 0.05 and ***p < 0.001 versus CON, one‐way ANOVA.
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