Osteocyte-derived extracellular vesicles mediate the bone-to-cartilage crosstalk and promote osteoarthritis progression

Abstract

Osteoarthritis is a common degenerative joint disease, in which mechanical overloading disrupts subchondral bone remodeling before cartilage degeneration and the osteocytes in the subchondral bone are mainly responsible for mechanosensing. However, their functional role in the early osteoarthritis is still unclear. Here we show that mechanical stress induces osteocytes in subchondral bone to secrete extracellular vesicles that accelerate cartilage metabolic dysregulation in patients with both sexes and male mice. The miR-23b-3p in extracellular vesicles promotes cartilage catabolism and inhibits anabolism by targeting OTUD4, disrupting mitophagy in chondrocytes. Inhibiting miR-23b-3p in osteocytes or chondrocytes reduces cartilage degeneration and osteoarthritis progression in male mice. Together, our findings highlight that osteocyte-derived extracellular vesicles mediate communication with chondrocytes and suggest miR-23b-3p as a potential therapeutic target for osteoarthritis.

Fig. 1. Extracellular vesicles facilitate crosstalk between osteocytes and chondrocytes in osteoarthritis progression.

A A schematic diagram illustrating the workflow utilizing primary human osteoarthritic chondrocytes co-cultured with osteocytes located in high stress (HS) and low stress (LS) region of the subchondral bone (SCB). B Levels of IL-1β, IL-6 and TNF-α measured by ELISA assays (n = 6). C The Alcian blue staining. Scale bar, 1 mm. D qPCR analysis of COL2A1, ACAN, SOX9, MMP13 and ADAMTS5 levels in primary human osteoarthritic chondrocytes co-cultured with LS/HS tissues (n = 3). E Western blot of COLII, ACAN, SOX9, MMP13 and ADAMTS5 protein expression in primary human osteoarthritic chondrocytes co-cultured with LS/HS tissues (n = 3). F RNA-sequencing and Gene Ontology (GO) enrichment analysis of the genes in LS/HS tissues. G A heat map (top) and its quantitation (bottom) indicating the expression of genes associated with extracellular vesicles (EVs) biogenesis within LS/HS tissues. H A schematic diagram summarizing the biology of EV biogenesis. I Representative electron microscopy images of intraluminal vesicles (ILVs) in LS/HS tissues. Scale bar, 500 nm. J Illustration of the animal experimental protocol for osteoarthritis (OA) modeling using osteocyte-specific ILVs/EVs reporter mice. K Safranin O staining. Scale bar, 200 μm. L The Osteoarthritis Research Society International (OARSI) scores (n = 6). M Localization of Cd63-GFP (green) in SCB osteocytes. Scale bar, 50 μm. SCB Subchondral bone, BM Bone marrow. N The semi-quantitative analysis of Cd63-GFP⁺ positive cells in SCB osteocytes (n = 6). O Localization of Cd63-GFP (green) in articular cartilage. The white arrows indicated Cd63-GFP (green). Scale bar, 50 μm. P The semi-quantitative analysis of Cd63-GFP⁺ positive cells in articular cartilage (n = 6). Data are presented as the mean ± SD. P values are from paired two-tailed Student’s t-test (B, D), Kruskal–Wallis test followed by two-tailed Mann–Whitney U test (L), one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (N, P) or Hypergeometric test (F). Source data are provided as a Source Data file.

Fig. 2. Osteocyte-derived extracellular vesicles promote chondrocyte catabolism and inhibit anabolism in vitro and in vivo.

A A schematic diagram illustrated that primary human osteoarthritic chondrocytes were treated with extracellular vesicles (EVs) extracted from low stress/high stress tissues (LS/HS-EVs). B The nanoparticle tracking analysis (NTA) analysis. C Representative confocal images showing the uptake of LS/HS-EVs in chondrocytes. Scale bar, 30 μm. D Alcian blue staining. Scale bar, 1 mm. E Western blot analysis of COLII, ACAN, SOX9, MMP13 and ADAMTS5 protein expression (n = 3). F A schematic diagram illustrating the administration of intra-articular injection of LS/HS-EVs to destabilization of the medial meniscus (DMM) mice. G Representative confocal images of PKH26-labeled osteocyte-EVs in the cartilage. Scale bar, 50 μm. H The Safranin O images (n = 8). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. I A schematic diagram illustrated that primary mouse chondrocytes were treated with Static/Load-EVs. J The NTA analysis. K Representative confocal fluorescence micrographs showing the uptake of EVs in chondrocytes. Scale bar, 30 μm. L Alcian blue staining. Scale bar, 2 mm. M Western blot analysis of ColII, Acan, Sox9, Mmp13 and Adamts5 protein level (n = 3). N A schematic diagram illustrating the administration of intra-articular injection of Static-EVs and Load-EVs to DMM mice. O Representative confocal images of PKH26-labeled MLO-Y4-EVs in the cartilage. Scale bar, 50 μm. P The Safranin O images of cartilage (n = 8). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. Q A schematic diagram illustrating the strategy for reducing osteocyte-derived EVs in DMM mice. R Representative immunohistochemical staining of Cd63 in subchondral bone (SCB) osteocytes (n = 8). Scale bars: 50 μm. S The Safranin O images (n = 8). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. Data are presented as the mean ± SD. Data was analyzed by unpaired two-tailed Student’s t-test (R). Source data are provided as a Source Data file.

Fig. 3. MiRNAs were responsible for osteocyte-derived extracellular vesicles in regulating cartilage metabolism.

A A schematic diagram illustrating the co-culturing system. Scale bar, 30 μm. B miR-223 expression in primary mouse chondrocytes (n = 3). C Images of primary mouse osteocytes isolated from wild-type (WT)- or osteocytes-specific Dicer knockout mice (cKO). Scale bar, 100 μm. D Western blot (left panel) and semi-quantitative analysis (right panel) of Dicer protein level in WT/cKO osteocytes (n = 3). E A schematic diagram illustrating the coculture of primary mouse chondrocytes and extracellular vesicles (EVs) from WT/cKO osteocytes subjected to mechanical loading (WT-EVs / cKO-EVs). F Alcian blue staining. Scale bar, 2 mm. G qPCR analysis of the Col2a1, Acan, Sox9, Mmp13 and Adamts5 levels in chondrocytes treated with WT-EVs or cKO-EVs (n = 3). H Western blot analysis (the left panel) and quantitative analysis (the right panel) of ColII, Acan, Sox9, Mmp13 and Adamts5 protein level in chondrocytes treated with WT-EVs or cKO-EVs (n = 3). I A schematic diagram showed that WT/cKO mice were performed Sham/destabilization of the medial meniscus (DMM) surgery. J Western blot (left panel) and semi-quantitative analysis (right panel) of Dicer protein level in cKO mice (n = 5). K Heat map of miRNA qPCR array (n = 3). L Safranin O images (n = 8). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. M The Osteoarthritis Research Society International (OARSI) scores (n = 8). N Representative confocal images of Acan, Sox9, Mmp13 and Adamts5 expression in cartilage from the indicated groups. Scale bars: 50 μm. O The quantitative analysis of Acan, Sox9, Mmp13 and Adamts5 protein (n = 8). Data are presented as the mean ± SD. P values are from unpaired two-tailed Student’s t-test (B, D, G, H and J), Kruskal–Wallis test followed by two-tailed Mann–Whitney U test (M), one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (O). Source data are provided as a Source Data file.

Fig. 4. MiR-23b-3p was the key mediator of osteocyte-derived extracellular vesicles in regulating cartilage metabolism.

A A schematic diagram illustrating the separation of extracellular vesicles (EVs) and their enrichment from human tissues and mouse plasma. Heatmaps of relative miRNA differences (B) and enrichment analysis (C). D The miR-23b-3p levels in LS-EVs and HS-EVs (n = 5). E The miR-23b-3p levels in subchondral bone (SCB) osteocytes from WT and Dicer-cKO mice with destabilization of the medial meniscus (DMM) surgery (n = 5). F The miR-23b-3p levels in cartilage and SCB osteocytes from human LS/HS tissues (n = 20). G The miR-23b-3p levels in cartilage and SCB osteocytes from mice with DMM surgery (n = 6). H The pre-miR-23b-3p levels in cartilage and SCB osteocytes from human LS/HS tissues (n = 20). I The pre-miR-23b-3p levels in cartilage and SCB osteocytes from mice with DMM surgery (n = 6). J Alcian blue staining. Scale bar in upper panel, 1 mm. Scale bar in lower panel, 2 mm. K Western blot analysis of COLII, ACAN, SOX9, MMP13 and ADAMTS5 protein expression in primary human and mouse chondrocytes transfected with nc or mimic (n = 3). L The schematic depicted the overexpression of miR-23b-3p in cartilage through intraarticular injection of rAAV5-miR-23b-3p-mcherry (rAAV5-miR-23b-3p) or negative control (rAAV5-nc) in 8-week-old male mice following DMM surgery (n = 8). M The Safranin O images of cartilage. Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. Data are presented as the mean ± SD. Data was analyzed by unpaired two-tailed Student’s t-test (D, E, G, I and K) and paired two-tailed Student’s t-test (F and H). Source data are provided as a Source Data file.

Fig. 5. Osteocyte-derived miR-23b-3p regulate cartilage metabolism during osteoarthritis progression.

A A schematic diagram illustrating the use of extracellular vesicles (EVs) derived from MLO-Y4 cells transfected with either a negative control (nc mimic-EVs) or a miR-23b-3p mimic (mimic-EVs), as well as EVs obtained from loaded MLO-Y4 cells transfected with either a negative control (nc inhibitor-EVs) or a miR-23b-3p inhibitor (inhibitor-EVs), which were subsequently added to primary mouse chondrocytes. B Alcian blue staining. Scale bar, 2 mm. C Western blot analysis of ColII, Acan, Sox9, Mmp13 and Adamts5 protein expression in primary mouse chondrocytes (n = 3). D qPCR analysis of the Col2a1, Acan, Sox9, Mmp13 and Adamts5 levels in primary mouse chondrocytes (n = 3). E A schematic diagram illustrating mouse study in which the Dmp1^cre mice were administered tail vein injection with rAAV9DSS₆-FLEx-miR-23b-3p (rAAV9DSS₆-miR-23b-3p) or rAAV9DSS₆-FLEx-nc (rAAV9DSS₆-nc) in destabilization of the medial meniscus (DMM) or sham surgery mice, to specifically overexpress miR-23b-3p expression in osteocytes (n = 7). F Safranin O images (n = 7). Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. G The Osteoarthritis Research Society International (OARSI) scores (n = 7). H A schematic diagram illustrating the mouse study in which Dmp1^cre mice were administered tail vein injection with rAAV9DSS₆-FLEx-miR-23b-3p-RNAi (rAAV9DSS₆-miR-23b-3p-RNAi) or rAAV9DSS₆-FLEx-nc (rAAV9DSS₆-nc) in DMM or sham surgery mice, to specifically antagonize miR-23b-3p expression in osteocytes (n = 7). I Safranin O images. Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. J The OARSI scores (n = 7). Data are presented as the mean ± SD. P values are from unpaired two-tailed Student’s t-test (D), Kruskal–Wallis test followed by two-tailed Mann–Whitney U test (G and J). Source data are provided as a Source Data file.

Fig. 6. MiR-23b-3p inhibits mitophagy by targeting Otud4 to skew the metabolism of chondrocytes.

A Gene Ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of potential targets for regulating cartilage metabolism from downregulated genes from human primary osteoarthritic chondrocytes transfected with negative control (nc) or miR-23b-3p mimics (mimic) using RNAseq. B Representative electron microscopy images of mitophagy in chondrocytes from low stress (LS) and high stress (HS) areas of clinical samples. Scale bars, 1 μm. C Mitophagy detection. Scale bars, 100 μm. D JC-1 staining. Scale bars, 50 μm. E Alcian blue staining. Scale bar, 1 mm. F Western blot analysis of PARKIN, PINK, MFN2, LC3B, DRP1, COLII, ACAN, SOX9, MMP13 and ADAMTS5 protein expression in human primary osteoarthritic chondrocytes (n = 3). G Venn diagram of miR-23b-3p targets. H qPCR analysis of OTUD4 levels in human samples (n = 12), Sham/destabilization of the medial meniscus (DMM) mice samples (n = 6), chondrocytes with nc/mimic transfection (n = 3) and chondrocytes with nc/inhibitor transfection (n = 3). I The representative confocal images of OTUD4 of cartilage in human samples (n = 10). Scale bars, 100 μm. J The representative confocal images of Otud4 of cartilage in Sham or DMM mice (n = 6). Scale bars, 50 μm. K Dual luciferase reporter activities after transfecting 239 T cells with miR-23b-3p and reporter carrying 3’ UTR in the long form of OTUD4 (n = 6). L Mitophagy detection. Scale bars, 100 μm. M JC-1 staining. Scale bars, 50 μm. N Alcian blue staining. Scale bar, 1 mm. O Western blot analysis of OTUD4, PARKIN, PINK, MFN2, LC3B, DRP1, COLII, ACAN, SOX9, MMP13 and ADAMTS5 protein expression in vehicle- and Lv-OTUD4-treated human primary osteoarthritic chondrocytes transfected with nc or mimic (n = 3). Data are presented as the mean ± SD. Data analyzed by paired two-tailed Student’s t-test (I), unpaired two-tailed Student’s t-test (H), one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (J and K) or Hypergeometric test (A). Source data are provided as a Source Data file.

Fig. 7. Therapeutic inhibition of miR-23b-3p in chondrocytes or osteocytes alleviated cartilage degeneration during osteoarthritis progression.

A A schematic diagram illustrating the intra-articular injection of antagomiR-23b-3p or antagomiR-nc in sham/destabilization of the medial meniscus (DMM) mice. Representative confocal images (B) and the quantitative analysis (C) of Otud4 in the articular cartilage (n = 7). Scale bar, 50 μm. D The qPCR analysis of Otud4 mRNA expression in chondrocytes (n = 3). E Representative electron microscopy images of mitophagy in chondrocytes. Scale bars, 500 nm. F The numbers of mitophagy in chondrocytes (n = 3). G Safranin O images. Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. H The Osteoarthritis Research Society International (OARSI) scores (n = 7). Representative confocal images (I) and the quantitative analysis (J) of Acan, Sox9, Mmp13 and Adamts5 in the articular cartilage. Scale bar, 50 μm. (n = 7). K A schematic diagram illustrating the workflow by which osteocyte-targeted antagomiR-23b-3p (LipoantagomiR-23b-3p@DSS₆) or nc (Liponc@DSS₆) was performed in DMM or sham mice. Representative confocal images (L) and the quantitative analysis (M) of Otud4 in the articular cartilage. Scale bar, 50 μm. (n = 7). N The qPCR analysis of Otud4 mRNA expression in the articular cartilage (n = 3). O Representative electron microscopy images of mitophagy in chondrocytes. Scale bars, 500 nm. P The numbers of mitophagy in chondrocytes (n = 3). Q Safranin O images. Scale bar in upper panel, 200 μm. Scale bar in lower panel, 100 μm. R The OARSI scores (n = 7). Representative confocal images (S) and the quantitative analysis (T) of Acan, Sox9, Mmp13 and Adamts5 in the articular cartilage (n = 7). Scale bar, 50 μm. Data are presented as the mean ± SD. P values are from Kruskal–Wallis test followed by two-tailed Mann–Whitney U test (H and R) and one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (C, D, F, J, M, N, P and T). Source data are provided as a Source Data file.

Fig. 8. Schematic of osteocyte-derived extracellular vesicles mediating bone-to-cartilage crosstalk and promoting osteoarthritis progression.

Created in BioRender. Shi (2025) https://BioRender.com/9xrn222.