Fig 1: Intra-articular T3-EV injection reversed OA progression by regulating the miR-455/SOX11/FOXO signaling axis.A To determine whether T3-EV transplantation could reduce or reverse the progression of OA, intra-articular injection of T3-EV labeled with Dir was performed for rats with DMM surgery. B PKH26-labeled EVs (red) to detect their internalization by BMSC in vitro. C Intra-articular delivery of the Dir-label T3-EVs (left: red) were monitored with in vivo fluorescence imaging to evaluate the near-infrared imaging effect and distribution of EVs for 12 weeks, showing an ideal intra-articular delivery effect (right). D–F Histological assessments of joint cartilage with D HE (left column), TB staining (right column), and E, F immunostaining for ACAN, MMP13, miR-455, SOX11, and FOXO1 in different groups. G–J Quantification and comparison of histological grade for OA progression in different groups (n = 8 for each). Joint destruction severity was determined with OARSI score, osteophyte formation, subchondral bone plate thickness, and synovial inflammation as previously reported60–62. Saline: OA model group with saline injection; un-EV: only untreated EVs were injected for OA treatment; T3-EV: TGFß3-treated EVs were injected for OA treatment. *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant. #p < 0.05 compared to the un-EV gel group.
Fig 2: Identification of T3-EV and un-EV.A Illustration of study design. B Representative images showing the morphology of T3-EV and un-EV visualized under transmission electron microscopy (TEM). C Particle size distribution of h T3-EVs measured using nanoparticle tracking Analysis (NTA). D Quantification of surface markers of EVs evaluated by western blotting. BMSCs served as the control in the western blot analysis of surface markers of EVs. E Representative fluorescence micrograph of PKH26 (red)-labeled EVs internalized by primary BMSCs. The labeled EVs were co-incubated with BMSCs for 24 h. F GAG staining with Safranin-O and Toluidine blue staining of BMSCs treated with EVs for 21 days. G Chondrogenesis was defined with immunostaining of SOX9, ACAN, and COL2A1(red). Counterstaining with F-actin (green) and DAPI (blue) was applied. H Three different experiments with the same BMSCs and the same EVs were performed. Chondrogenic gene expression (n = 3 for each) was assayed with qRT-PCR for SOX9, ACAN, and COL2A1. I–K Quantification of deposited GAGs and collagens (n = 3 for each) was also performed with the same BMSCs and the same EVs to demonstrate the chondrogenic lineage committed by the BMSCs. Treatment with saline served as control. *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant.
Fig 3: MiR-455 regulates chondrogenesis and OA development by modulating the SOX11/FOXO signaling pathway.A Schematic representation of how the miR-455/SOX11/FOXO signaling pathway might mediate chondrogenesis and the therapeutic effects of T3-EV in OA treatment and cartilage regeneration. B Cultured primary human BMSCs were transfected with miR-455 mimics, miR-455 inhibitor, their negative controls, control siRNA or SOX11 siRNA for 72 h, respectively, and the expression levels of chondrogenic markers SOX9, ACAN, COL2A1, MMP13, and SOX11/FOXO signaling pathway markers SOX11, FOXO1, Gadd45a, p27, and Cathepsin L were assessed with western blot. C Rescue experiments was established in cultured primary BMSCs to validate the relationship between miR-455 and SOX11. Elevation of SOX9 and ACAN expression levels by miR-455 mimics was rescued by restoration of SOX11 expression. In comparison, inhibition of SOX9, ACAN, and FOXO1 expression by SOX11 overexpression was rescued by miR-455 mimics. D Upregulation of SOX9 and ACAN expression levels by SOX11 siRNA was abolished by silencing of FOXO1 expression. In comparison, upregulation of FOXO1 target genes (p27 and Cathepsin L) expression by FOXO1 siRNA was abolished by silencing of SOX11 expression.
Fig 4: Activation of FOXO signaling by T3-EV.A Heatmap of clustering dysregulated mRNA expression profiles with microarray in T3-EV-treated BMSCs compared to un-EV treated control. B Volcano plot of mRNA expression profiles in T3-EV-treated BMSC recipient. C Dysregulated typical chondrogenic markers derived from the microarray results with T3-EV treatment. D All differentially expressed genes were subjected to gene ontology (GO) analysis (DEG with fold change >2 or <0.5, p value <0.01). BP biological processes, MF molecular function, CC cellular component. E Significantly enriched pathways for dysregulated genes enriched with T3-EV treatment in KEGG pathways. F, G FOXO1 expression(red) assayed with western blotting and fluorescent immunostaining (red for FOXO1; green for cytoskeleton) in T3-EV treated BMSC recipient. Cells were counterstained with DAPI for the nucleus (blue). Treatment with saline served as control. H Quantification of gene expression with the same BMSCs and the same EVs (n = 3 for each) with qRT-PCR for Chondrogenic genes (SOX9, ACAN, COL2A1, and MMP13) and FOXO signaling-related genes (FOXO1, Gadd45a, p27, and Cathepsin L). *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant.
Supplier Page from Biomatik for Human COL2 (Collagen Type Ⅱ) ELISA Kit