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Cited by 17Osteoarthritis Treatment and MechanismsMesenchymal stem cell researchExtracellular vesicles in disease

The DOI matches its Crossref record; 85 of the 86 references it cites were found in a registry or an index.

Functional and Molecular Analysis of Human Osteoarthritic Chondrocytes Treated with Bone Marrow-Derived MSC-EVs

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Scalzone, Annachiara, Sanjurjo-Rodríguez, Clara, Berlinguer-Palmini, Rolando, Dickinson, Anne M., Jones, Elena, Wang, Xiao-Nong, Crossland, Rachel E.·2024·Bioengineering
DOI 10.3390/bioengineering11040388PMC PMC11047960
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Abstract

Osteoarthritis (OA) is a degenerative joint disease, causing impaired mobility. There are currently no effective therapies other than palliative treatment. Mesenchymal stromal cells (MSCs) and their secreted extracellular vesicles (MSC-EVs) have shown promise in attenuating OA progression, promoting chondral regeneration, and modulating joint inflammation. However, the precise molecular mechanism of action driving their beneficial effects has not been fully elucidated. In this study, we analyzed MSC-EV-treated human OA chondrocytes (OACs) to assess viability, proliferation, migration, cytokine and catabolic protein expression, and microRNA and mRNA profiles. We observed that MSC-EV-treated OACs displayed increased metabolic activity, proliferation, and migration compared to the controls. They produced decreased proinflammatory (Il-8 and IFN-γ) and increased anti-inflammatory (IL-13) cytokines, and lower levels of MMP13 protein coupled with reduced expression of MMP13 mRNA, as well as negative microRNA regulators of chondrogenesis (miR-145-5p and miR-21-5p). In 3D models, MSC-EV-treated OACs exhibited enhanced chondrogenesis-promoting features (elevated sGAG, ACAN, and aggrecan). MSC-EV treatment also reversed the pathological impact of IL-1β on chondrogenic gene expression and extracellular matrix component (ECM) production. Finally, MSC-EV-treated OACs demonstrated the enhanced expression of genes associated with cartilage function, collagen biosynthesis, and ECM organization and exhibited a signature of 24 differentially expressed microRNAs, associated with chondrogenesis-associated pathways and ECM interactions. In conclusion, our data provide new insights on the potential mechanism of action of MSC-EVs as a treatment option for early-stage OA, including transcriptomic analysis of MSC-EV-treated OA, which may pave the way for more targeted novel therapeutics.

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Figures

Figure 1 Characteristics of MSCs and MSC-EVs. (A) MSC morphology and tri-lineage potential (adipogenesis, osteogenesis, chondrogenesis) assessment. (B) FC histogram showing MSC surface phenotype profiles for lineage specific markers (CD14, CD19, CD34, CD45), HLA-DR, CD105, CD73, and CD90. (C) TEM image showing EV morphology. (D FC histogram showing positive expression of EV markers CD63, CD81, and CD9. (E) NTA analysis showing EV size distribution. (F) Western blot analysis showing positive expression of EV markers Flotillin-1 and Alix in three independent MSC-EV examples (S1 to S3).
Figure 1 Characteristics of MSCs and MSC-EVs. (A) MSC morphology and tri-lineage potential (adipogenesis, osteogenesis, chondrogenesis) assessment. (B) FC histogram showing MSC surface phenotype profiles for lineage specific markers (CD14, CD19, CD34, CD45), HLA-DR, CD105, CD73, and CD90. (C) TEM image showing EV morphology. (D FC histogram showing positive expression of EV markers CD63, CD81, and CD9. (E) NTA analysis showing EV size distribution. (F) Western blot analysis showing positive expression of EV markers Flotillin-1 and Alix in three independent MSC-EV examples (S1 to S3).CC BY · PubMed Central
Figure 2 MSC-EV-treated OACs’ EV uptake, viability, mobility, and migration. (A) Confocal microscopy images to show MSC-EV-treated OACs. OACs internalize MSC-EVs after 1 h of co-incubation. Cut view confirms internalization of MSC-EVs. (B) Analysis of metabolic activity of OACs and EV-treated OACs (OAC + EV) via CellTiter-Glo 2.0 assay after 5 days of culture. Significance was calculated using the Wilcoxon matched pairs signed-rank test. (C) Two-dimensional scratch assay: example images for OAC and OAC + EV samples at 0 and 40 h post-scratch. Significance was calculated using a mixed-effects model, with Sidak’s multiple comparisons test. (D) Cell migration assay: example images are shown for OAC and OAC + EV samples, assessed using a transwell filter system. Total cell count is shown for OAC and OAC ± EV. Significance was calculated using the paired t-test. (B–D) Lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.
Figure 2 MSC-EV-treated OACs’ EV uptake, viability, mobility, and migration. (A) Confocal microscopy images to show MSC-EV-treated OACs. OACs internalize MSC-EVs after 1 h of co-incubation. Cut view confirms internalization of MSC-EVs. (B) Analysis of metabolic activity of OACs and EV-treated OACs (OAC + EV) via CellTiter-Glo 2.0 assay after 5 days of culture. Significance was calculated using the Wilcoxon matched pairs signed-rank test. (C) Two-dimensional scratch assay: example images for OAC and OAC + EV samples at 0 and 40 h post-scratch. Significance was calculated using a mixed-effects model, with Sidak’s multiple comparisons test. (D) Cell migration assay: example images are shown for OAC and OAC + EV samples, assessed using a transwell filter system. Total cell count is shown for OAC and OAC ± EV. Significance was calculated using the paired t-test. (B–D) Lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.CC BY · PubMed Central
Figure 3 MSC-EV-treated OACs’ cytokine, catabolic protein, gene, and microRNA expression. (A) Secreted cytokine production (IFN-γ, IL-8, IL-13) by MSC-EV-treated OACs by multiplex immunoassay in n = 12 OAC and OAC + EV. (B) Secreted MMP13 protein production by ELISA in n = 15 OAC and OAC + EV, and gene expression assessed by qRT-PCR in 2D monolayer cultures in n = 11 matched OAC controls and OAC + EV. (C) MicroRNA expression assessed by qRT-PCR in 2D monolayer cultures in n = 11 matched OAC controls and OAC + EV. (A–C) Expression differences between groups were calculated using the paired t-test, and lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.
Figure 3 MSC-EV-treated OACs’ cytokine, catabolic protein, gene, and microRNA expression. (A) Secreted cytokine production (IFN-γ, IL-8, IL-13) by MSC-EV-treated OACs by multiplex immunoassay in n = 12 OAC and OAC + EV. (B) Secreted MMP13 protein production by ELISA in n = 15 OAC and OAC + EV, and gene expression assessed by qRT-PCR in 2D monolayer cultures in n = 11 matched OAC controls and OAC + EV. (C) MicroRNA expression assessed by qRT-PCR in 2D monolayer cultures in n = 11 matched OAC controls and OAC + EV. (A–C) Expression differences between groups were calculated using the paired t-test, and lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.CC BY · PubMed Central
Figure 4 Chondrogenic potential of MSC-EV-treated OACs in a 3D spheroid model. (A) Quantification of sGAG production in OAC and OAC + EV samples. (B) Representative histological analysis of toluidine blue, which stains sGAGs, on tissue slices from three independent experiments (scale bar = 200 µm). Zoomed image is shown in the center and in the peripheral zone of the spheroid slice (scale bar = 125 µm). (C) ACAN relative expression via RT-qPCR, for OAC and OAC + EV, at day 21 with respect to day 2. (D) Representative IHC of aggrecan on tissue slices for OAC and OAC + EV samples (scale bar = 200 µm). Zoomed image is shown in the center and in the peripheral zone of the spheroid slice (scale bar = 125 µm). (A,C) Significance was calculated using the paired t-test and lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.
Figure 4 Chondrogenic potential of MSC-EV-treated OACs in a 3D spheroid model. (A) Quantification of sGAG production in OAC and OAC + EV samples. (B) Representative histological analysis of toluidine blue, which stains sGAGs, on tissue slices from three independent experiments (scale bar = 200 µm). Zoomed image is shown in the center and in the peripheral zone of the spheroid slice (scale bar = 125 µm). (C) ACAN relative expression via RT-qPCR, for OAC and OAC + EV, at day 21 with respect to day 2. (D) Representative IHC of aggrecan on tissue slices for OAC and OAC + EV samples (scale bar = 200 µm). Zoomed image is shown in the center and in the peripheral zone of the spheroid slice (scale bar = 125 µm). (A,C) Significance was calculated using the paired t-test and lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.CC BY · PubMed Central
Figure 5 Effect of MSC-EVs and IL-1β on OAC chondrogenic gene expression and ECM component. (A) Relative gene expression of ACAN, SOX9, COL2A1, COL10A1, MMP13, and ADAMTS at day 10 with respect to day 2. (B) Quantification of sGAG production in digested spheroids formed from OAC, OAC + IL, and OAC + IL + EV samples. (C) Representative histological analysis of Toluidine blue staining GAGs from three independent experiments. Scale bars represent 200 µm and 125 µm, respectively, for original and zoomed-in areas, as indicated. (D) Representative IHC of aggrecan staining on tissue sections of OAC, OAC + IL, and OAC + IL + EV samples from three independent experiments. Scale bars represent 200 µm and 125 µm, respectively, for original and zoomed areas, as indicated. (A,B) Significance was calculated using one-way ANOVA and lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.
Figure 5 Effect of MSC-EVs and IL-1β on OAC chondrogenic gene expression and ECM component. (A) Relative gene expression of ACAN, SOX9, COL2A1, COL10A1, MMP13, and ADAMTS at day 10 with respect to day 2. (B) Quantification of sGAG production in digested spheroids formed from OAC, OAC + IL, and OAC + IL + EV samples. (C) Representative histological analysis of Toluidine blue staining GAGs from three independent experiments. Scale bars represent 200 µm and 125 µm, respectively, for original and zoomed-in areas, as indicated. (D) Representative IHC of aggrecan staining on tissue sections of OAC, OAC + IL, and OAC + IL + EV samples from three independent experiments. Scale bars represent 200 µm and 125 µm, respectively, for original and zoomed areas, as indicated. (A,B) Significance was calculated using one-way ANOVA and lines represent mean ± SEM. The significance threshold for p-values was p < 0.05.CC BY · PubMed Central
Figure 6 Gene expression profile of MSC-EV-treated OACs compared to OACs. (A) Heatmap shows unsupervised hierarchical clustering of 54 genes that were significantly differentially expressed between groups: each column represents an individual sample. OAC + EV are depicted in blue, while control OACs are depicted in pink. Patient gender (green, purple) and age (green scale) are also indicated. Relative expression changes are indicated by the color scale (red: high; blue: low). (B) Principal component analysis of MSC-EV-treated OACs vs. control OACs. (C) Reactome pathway analysis for genes significantly differentially expressed between OAC + EV and OAC controls. Both analyses indicate the number of genes implicated in each pathway.
Figure 6 Gene expression profile of MSC-EV-treated OACs compared to OACs. (A) Heatmap shows unsupervised hierarchical clustering of 54 genes that were significantly differentially expressed between groups: each column represents an individual sample. OAC + EV are depicted in blue, while control OACs are depicted in pink. Patient gender (green, purple) and age (green scale) are also indicated. Relative expression changes are indicated by the color scale (red: high; blue: low). (B) Principal component analysis of MSC-EV-treated OACs vs. control OACs. (C) Reactome pathway analysis for genes significantly differentially expressed between OAC + EV and OAC controls. Both analyses indicate the number of genes implicated in each pathway.CC BY · PubMed Central
Figure 7 Differential microRNA expression profiles of OAC + EV and OAC. (A) NanoString microRNA expression profiling: heatmap shows unsupervised hierarchical clustering of significantly differentially expressed microRNAs (p < 0.05, n = 24), based on normalized expression counts, in OAC + EV and controls. Each column represents an individual sample. Relative expression changes are indicated by the color scale (red: high; blue: low). OAC + EV are depicted by black shading, while OAC are depicted by grey shading. (B) KEGG pathways analysis for the 24 differentially expressed microRNAs in OAC + EV vs. control OACs. Pathway analysis was performed using Diana Tools miRPath, based on TarBase v8.0 and the number of target genes and targeting microRNAs are plotted for each pathway (FDR-corrected p-values < 0.05). (C) Validation of selected microRNA expression in an independent cohort of OAC + EV and OAC controls (n = 11) by qRT-PCR. Lines represent mean ± SEM. Significance was calculated using the paired t-test, and the threshold for p-values was p < 0.05. Heatmap to show unsupervised hierarchical clustering of the same microRNAs according to NanoString data (n = 7 matched samples and controls).
Figure 7 Differential microRNA expression profiles of OAC + EV and OAC. (A) NanoString microRNA expression profiling: heatmap shows unsupervised hierarchical clustering of significantly differentially expressed microRNAs (p < 0.05, n = 24), based on normalized expression counts, in OAC + EV and controls. Each column represents an individual sample. Relative expression changes are indicated by the color scale (red: high; blue: low). OAC + EV are depicted by black shading, while OAC are depicted by grey shading. (B) KEGG pathways analysis for the 24 differentially expressed microRNAs in OAC + EV vs. control OACs. Pathway analysis was performed using Diana Tools miRPath, based on TarBase v8.0 and the number of target genes and targeting microRNAs are plotted for each pathway (FDR-corrected p-values < 0.05). (C) Validation of selected microRNA expression in an independent cohort of OAC + EV and OAC controls (n = 11) by qRT-PCR. Lines represent mean ± SEM. Significance was calculated using the paired t-test, and the threshold for p-values was p < 0.05. Heatmap to show unsupervised hierarchical clustering of the same microRNAs according to NanoString data (n = 7 matched samples and controls).CC BY · PubMed Central

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  1. Théry, C., Amigorena, S., Raposo, G., and Clayton, A. (2006). Current Protocols in Cell Biology, Wiley. Chapter 3, Unit 3.22.

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Metadata

Authors
Scalzone, A., Sanjurjo-Rodríguez, C., Berlinguer-Palmini, R., Dickinson, A. M., Jones, E., Wang, X.-N., & Crossland, R. E.
Affiliations
  • Italian Institute of Technology (IT)
  • Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds LS7 4SA, UK
  • Bioimaging Unit, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK
  • Newcastle University (GB)
  • University of Leeds (GB)
  • Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK
Corresponding author
Crossland, Rachel E.
ORCID
Venue
Bioengineering
Publisher
MDPI AG
Published
2024-04-17
Volume
11
Issue
4
Pages
388
ISSN
2306-5354 (ISSN-L)
DOI
10.3390/bioengineering11040388
PMC
PMC11047960
OpenAlex
W4394915029
Cited by
16
References
86
Citations by year
2026: 4 · 2025: 10 · 2024: 2
FWCI
5.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Bioengineering (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Newcastle University ePrints (Newcastle Univesity) (cc-by)
Funders
Tissue Engineering and Regenerative Therapies Centre Versus Arthritis (21156)
Topics
  • Health Sciences › Medicine › Rheumatology › Osteoarthritis Treatment and Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Mesenchymal stem cell research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
Keywords
osteoarthritis, chondrocytes, chondrogenesis, extracellular matrix, microRNA, MMP-13, IL-1β, cytokine expression, cartilage regeneration, gene expression profiling

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