Journal: Materials Today Bio

Article Title: EXO/hydrogel system for sequential regulation of endogenous hyaline cartilage regeneration

doi: 10.1016/j.mtbio.2025.102598

Figure Lengend Snippet: Effects of the Gel-C-E H + E K hydrogel on the differentiation and migration capacity of BMSCs. (A) Expression changes of chondrogenic-related proteins in BMSCs chondrogenically induced on different hydrogels for 21 days. (B) Expression changes of chondrogenic-related genes in BMSCs chondrogenically induced on different hydrogels for 21 days. (C) Scratch wound healing images of BMSCs in different hydrogels at 24 h. (D) Analysis of scratch wound healing results of BMSCs in different hydrogels at 24 h. (E) Analysis of Transwell migration results of BMSCs in different hydrogels at 24 h. (F) Transwell migration images of BMSCs in different hydrogels at 24 h. (G) Alcian blue staining of BMSCs after 21 days of chondrogenic induction on different hydrogels. (H) Histological staining (H&E, Alcian blue, Toluidine blue) of BMSCs spheroids cultured in different hydrogels. Data are presented as mean ± SD, ns = not significant, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: The Gel-C-E H + E K hydrogel demonstrated optimal chondrogenic differentiation efficacy ( ) To better simulate the in vivo chondrogenic microenvironment and ensure a consistent inductive background across all experimental groups, all subsequent groups—including Ctrl, Gel-C-E, Gel-C-E H , and Gel-C-E H + E K groups—were cultured in the standard chondrogenic induction medium (G417, Servicebio).

Techniques: Migration, Expressing, Staining, Cell Culture

Journal: Materials Today Bio

Article Title: EXO/hydrogel system for sequential regulation of endogenous hyaline cartilage regeneration

doi: 10.1016/j.mtbio.2025.102598

Figure Lengend Snippet: Positive effect of the Gel-C-E H + E K hydrogel on maintaining the stable chondrocytic phenotype. (A) IF observation of COL1 and COL2 expression in RCs seeded in different hydrogels under stimulation with 50 ng/mL IL-1β. (B) Quantification of COL2 fluorescence intensity from immunofluorescence images. (C) Quantification of COL1 fluorescence intensity from immunofluorescence images. (D) Expression levels of chondrogenic marker proteins in RCs seeded in different hydrogels under normal culture conditions (without inflammatory stimulation). (E) Changes in chondrogenic marker protein expression levels in chondrocytes seeded in different hydrogels under inflammatory stimulation. (F) Gene expression of chondrogenesis markers in chondrocytes seeded in different hydrogels under normal culture and inflammatory stimulation conditions. (G) DCFH-DA fluorescence staining for assessing the ROS scavenging effects of different hydrogels in RCs. (H) Fluorescence images of ROS levels in different hydrogels after DCFH-DA staining. (I) Flow cytometry assessment of ROS levels in RCs exposed to different hydrogels, evaluating ROS scavenging efficiency. (J) Flow cytometry analysis of ROS levels in RCs exposed to different hydrogels. Data are presented as mean ± SD, ns = not significant, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: The Gel-C-E H + E K hydrogel demonstrated optimal chondrogenic differentiation efficacy ( ) To better simulate the in vivo chondrogenic microenvironment and ensure a consistent inductive background across all experimental groups, all subsequent groups—including Ctrl, Gel-C-E, Gel-C-E H , and Gel-C-E H + E K groups—were cultured in the standard chondrogenic induction medium (G417, Servicebio).

Techniques: Expressing, Fluorescence, Immunofluorescence, Marker, Gene Expression, Staining, Flow Cytometry

Journal: Materials Today Bio

Article Title: EXO/hydrogel system for sequential regulation of endogenous hyaline cartilage regeneration

doi: 10.1016/j.mtbio.2025.102598

Figure Lengend Snippet: Exploration of the anti-inflammatory mechanism of the Gel-C-E H + E K hydrogel system. (A) Release levels of inflammatory cytokines under normal and inflammatory conditions, measured by Elisa. (B) Influence of HF and KGN on the gene expression levels of inflammatory cytokines under normal and inflammatory conditions. (C) Gene expression levels of inflammatory cytokines under normal and inflammatory conditions. (D) Influence of HF and KGN on the expression levels of chondrogenic marker proteins under normal and inflammatory conditions. (E) WB images validating the affinity between HF and cGAS using CETSA. (F) Thermal shift images validating the affinity between HF and cGAS using CETSA. (G) Positive influence of HF on chondrogenic marker protein expression under inflammatory conditions. (H) Co-IP further confirms the binding affinity between HF and cGAS. (I) Downstream signaling cascade following HF-cGAS binding. (J) Schematic diagram illustrating the immunomodulatory mechanism of HF via binding to cGAS. Data are presented as mean ± SD, ns = not significant, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Article Snippet: The Gel-C-E H + E K hydrogel demonstrated optimal chondrogenic differentiation efficacy ( ) To better simulate the in vivo chondrogenic microenvironment and ensure a consistent inductive background across all experimental groups, all subsequent groups—including Ctrl, Gel-C-E, Gel-C-E H , and Gel-C-E H + E K groups—were cultured in the standard chondrogenic induction medium (G417, Servicebio).

Techniques: Enzyme-linked Immunosorbent Assay, Gene Expression, Expressing, Marker, Co-Immunoprecipitation Assay, Binding Assay

Journal: Bone & Joint Research

Article Title: Wogonin pretreatment of infrapatellar fat pad mesenchymal stem cell-derived exosomes advances articular cartilage repair in osteoarthritis

doi: 10.1302/2046-3758.1412.BJR-2024-0316.R2

Figure Lengend Snippet: Characterization of human primary chondrocytes and infrapatellar fat pad mesenchymal stem cells (MSCs IPFP ). Human primary chondrocytes were isolated and extracted: a) the microscope (scale bar: 100 μm) was used to observe cell morphology; b) Toluidine blue staining (scale bar: 50 μm) was applied to stain the proteoglycans in the cytoplasm of chondrocytes in purple; c) type II collagen immunofluorescence staining and 4',6-diamidino-2-phenylindole (DAPI) immunofluorescence staining were combined, and chondrocyte nuclei were stained blue by DAPI, whereas type II collagen was stained green (scale bar, 20 μm). Human IPFP-derived cells were isolated and extracted: d) the microscope (scale bar: 100 μm) was applied to observe cell morphology; e) CD29, CD44, CD90, CD31, CD117, and CD45 expression was tested by flow cytometry; f) adipogenic, chondrogenic, and osteogenic differentiation assays (scale bar: 100 μm).

Article Snippet: MSCs IPFP after three weeks of induction in a chondrogenic induction medium (OriCell) were stained using an Alcian Blue Staining Kit (G1565; Solarbio).

Techniques: Isolation, Microscopy, Staining, Immunofluorescence, Derivative Assay, Expressing, Flow Cytometry

Journal: Bioactive Materials

Article Title: 3D-printed advanced scaffold armed with exosomes derived from human skeletal stem cell identified by single-cell RNA sequencing enhances osteochondral regeneration

doi: 10.1016/j.bioactmat.2025.04.028

Figure Lengend Snippet: Schematic illustration of the construction of a biomimetic 3D-printed scaffold armed with SSC-Exos for osteochondral regeneration. (A) Identification and isolation of a novel SSC population from the IFP using single-cell sequencing and flow cytometry, followed by the extraction of their Exos. (B) Fabrication of SSC-Exos-loaded 3D-printed hydrogel scaffolds using advanced 3D printing technology and in situ transplantation of the biomimetic scaffolds at osteochondral defect sites, promoting simultaneous regeneration of cartilage and subchondral bone. (C) MiR-214-3p, released by SSC-Exos, enhances the chondrogenic differentiation of BMSCs, highlighting the molecular mechanism underlying cartilage regeneration.

Article Snippet: For chondrogenic differentiation, BMSCs or SSCs were cultured in chondrogenic induction medium (DMEM supplemented with 0.1 μmol/L dexamethasone, 1.25 mg/mL bovine serum albumin, 1 mmol/L sodium pyruvate, ITS, 10 ng/mL TGF-β, 37.5 μg/mL vitamin C, and 1 ng/mL β-FGF) (Cyagen, China) for 21 days and observed after staining with chondrogenic staining solution (Cyagen, China).

Techniques: Isolation, Sequencing, Flow Cytometry, Extraction, In Situ, Transplantation Assay

Journal: Bioactive Materials

Article Title: 3D-printed advanced scaffold armed with exosomes derived from human skeletal stem cell identified by single-cell RNA sequencing enhances osteochondral regeneration

doi: 10.1016/j.bioactmat.2025.04.028

Figure Lengend Snippet: Identification of SSC Subpopulations by Single-Cell RNA Sequencing. (A) Schematic illustration of the ScRNA-seq workflow.(B) UMAP plot displaying clusters and annotated cell types. (C) Expression levels of marker genes for different cell types across clusters. (D) UMAP plot showing the expression levels of SSC markers in various clusters. (E) Bubble plot illustrating differences in marker expression between SSCs and other ASPCs. (F) UMAP plot highlighting the SSC subcluster isolated from the broader ASPC population, as indicated by the color-coded legend. (G) Relative contribution of each ASPC phenotype in the IFP versus SAT. (H) Predicted differentiation state scores of SSCs compared to other ASPCs, as estimated by CytoTRACE. (I) UMAP plot depicting the differentiation state of SSCs within the total ASPC population, as estimated by CytoTRACE. (J) UMAP plot showing PDGFRA expression levels in the SSC cluster among all ASPCs. (K) UMAP plot distinguishing SSCs derived from the IFP and SAT, as indicated by the color-coded legend. (L) UMAP plot illustrating the differentiation state of SSCs from the IFP and SAT, as estimated by CytoTRACE. (M) Predicted differentiation state scores of SSCs from the IFP versus SAT, as estimated by CytoTRACE. (N) UMAP plot showing PDGFRA expression levels in SSCs derived from the IFP and SAT. (O) Heatmap of pathway activity scores for chondrogenic-related pathways in SSCs from the IFP and SAT. (P) UMAP plot displaying subclusters stratified from the ASPC population of the IFP. (Q) UMAP plot showing the differentiation state of SSCs within the IFP, as estimated by CytoTRACE. (R) Predicted differentiation state scores of SSCs within the IFP, as estimated by CytoTRACE. (S) UMAP plot illustrating PDGFRA expression levels in the SSC cluster within the IFP. (T) Enriched GO terms associated with chondrogenic differentiation in SSCs. The bar chart indicates the number of genes enriched in each term.Bar chart shows the number of genes enriched in each term.

Article Snippet: For chondrogenic differentiation, BMSCs or SSCs were cultured in chondrogenic induction medium (DMEM supplemented with 0.1 μmol/L dexamethasone, 1.25 mg/mL bovine serum albumin, 1 mmol/L sodium pyruvate, ITS, 10 ng/mL TGF-β, 37.5 μg/mL vitamin C, and 1 ng/mL β-FGF) (Cyagen, China) for 21 days and observed after staining with chondrogenic staining solution (Cyagen, China).

Techniques: RNA Sequencing, Expressing, Marker, Isolation, Derivative Assay, Activity Assay

Journal: Bioactive Materials

Article Title: 3D-printed advanced scaffold armed with exosomes derived from human skeletal stem cell identified by single-cell RNA sequencing enhances osteochondral regeneration

doi: 10.1016/j.bioactmat.2025.04.028

Figure Lengend Snippet: Chondrogenic properties of SSC-Exos in vitro . (A) Representative immunofluorescence images and 3D reconstructions showing the uptake of Exos (PKH26-labeled, red) by BMSCs (phalloidin-labeled, green). Nuclei are counterstained with DAPI (blue). Scale bar: 10 μm. (B) Representative immunofluorescence images and (G) quantitative results showing the expression of SOX9 (green) in BMSCs co-cultured with different Exos. Each group n = 3. Scale bar: 50 μm; enlarged view scale bar: 3 μm. (C) Alcian Blue staining of BMSCs co-cultured with different Exos. Scale bar: 10 μm. (D) Representative immunofluorescence images of COL2 (red) and DAPI (blue) staining in chondrocytes treated with SSC-Exos. Scale bar: 20 μm. (E) Representative immunofluorescence images of ACAN (red) and DAPI (blue) staining in chondrocytes treated with SSC-Exos. Quantitative results are shown in .

Article Snippet: For chondrogenic differentiation, BMSCs or SSCs were cultured in chondrogenic induction medium (DMEM supplemented with 0.1 μmol/L dexamethasone, 1.25 mg/mL bovine serum albumin, 1 mmol/L sodium pyruvate, ITS, 10 ng/mL TGF-β, 37.5 μg/mL vitamin C, and 1 ng/mL β-FGF) (Cyagen, China) for 21 days and observed after staining with chondrogenic staining solution (Cyagen, China).

Techniques: In Vitro, Immunofluorescence, Labeling, Expressing, Cell Culture, Staining

Journal: Bioactive Materials

Article Title: 3D-printed advanced scaffold armed with exosomes derived from human skeletal stem cell identified by single-cell RNA sequencing enhances osteochondral regeneration

doi: 10.1016/j.bioactmat.2025.04.028

Figure Lengend Snippet: MiR-214-3p Mediates the Effect of SSC-Exos on Promoting Chondrogenic Differentiation of BMSCs. (A) Heatmap of differentially expressed miRNAs in SSC-Exos and ADSC-Exos. Red and blue colors represent upregulated and downregulated expressions, respectively. (B) Volcano plot of miRNAs differentially expressed between SSC-Exos and ADSC-Exos. (C) qRT-PCR quantification of the top 10 significantly expressed miRNAs (Log2FC > 1) (normalized to GAPDH) between SSC-Exos and ADSC-Exos. Each group n = 6. (D) Representative images of Alcian Blue staining of BMSCs co-cultured with PBS, SSC-Exos, miR-214-3p mimic, and miR-214-3pIN-Exos. Scale bar: 20 μm. (E) Representative immunofluorescence images of SOX9 (green) in BMSCs co-cultured with PBS, SSC-Exos, miR-214-3p mimic, and miR-214-3pIN-Exos. Scale bar: 50 μm. (F, G) Quantification of Alcian Blue staining results shown in (D). (H) Quantitative results of SOX9 expression from (E). (I) Quantification of chondrogenesis-related genes in BMSCs co-cultured with PBS, SSC-Exos, miR-214-3p mimic, and miR-214-3pIN-Exos. Each group n = 3. Statistical significance was calculated using one-way ANOVA with Tukey's post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: For chondrogenic differentiation, BMSCs or SSCs were cultured in chondrogenic induction medium (DMEM supplemented with 0.1 μmol/L dexamethasone, 1.25 mg/mL bovine serum albumin, 1 mmol/L sodium pyruvate, ITS, 10 ng/mL TGF-β, 37.5 μg/mL vitamin C, and 1 ng/mL β-FGF) (Cyagen, China) for 21 days and observed after staining with chondrogenic staining solution (Cyagen, China).

Techniques: Quantitative RT-PCR, Staining, Cell Culture, Immunofluorescence, Expressing

Journal: Bioactive Materials

Article Title: 3D-printed advanced scaffold armed with exosomes derived from human skeletal stem cell identified by single-cell RNA sequencing enhances osteochondral regeneration

doi: 10.1016/j.bioactmat.2025.04.028

Figure Lengend Snippet: JAG2 overexpression attenuates the effect of SSC-Exos in promoting BMSC chondrogenic differentiation. (A) Venn diagram demonstrated the potential target genes of miR-214-3p. (B) Quantification of JAG2 (normalized to GAPDH) of the target gene miR-214-3p in BMSCs treated with PBS or SSC-Exos. Each group n = 6. (C) Luciferase reporter assay to confirm that JAG2 is a target gene for miR-214-3p. (D) Western blot of JAG2 in BMSCs treated with PBS, SSC-Exos, and miR-214-3p IN -Exos. Each group n = 3. (E) Quantification of JAG2 relative protein expression (normalized to GAPDH) in (D). (F) Representative images of Alcian blue staining of BMSCs co-cultured with PBS, SSC-Exos + Vector and SSC-Exos + JAG2 Plasmid. Scale bar: 20 μm. (G) Representative immunofluorescence images of SOX9 (green) in BMSCs co-cultured with PBS,SSC-Exos + Vector and SSC-Exos + JAG2 Plasmid. Scale bar: 50 μm. (H, I) Quantification of Alcian blue staining results shown in (F). Each group n = 3. (J) Quantification of SOX9 immunofluorescence intensity in (G). Each group n = 6. (K) Quantification of chondrogenesis related genes in BMSCs co-cultured with PBS, SSC-Exos + Vector and SSC-Exos + JAG2 Plasmid. Each group n = 3. Data are shown as the mean ± SD (n ≥ 3). Statistical significance was calculated using one-way ANOVA with Tukey's post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Article Snippet: For chondrogenic differentiation, BMSCs or SSCs were cultured in chondrogenic induction medium (DMEM supplemented with 0.1 μmol/L dexamethasone, 1.25 mg/mL bovine serum albumin, 1 mmol/L sodium pyruvate, ITS, 10 ng/mL TGF-β, 37.5 μg/mL vitamin C, and 1 ng/mL β-FGF) (Cyagen, China) for 21 days and observed after staining with chondrogenic staining solution (Cyagen, China).

Techniques: Over Expression, Luciferase, Reporter Assay, Western Blot, Expressing, Staining, Cell Culture, Plasmid Preparation, Immunofluorescence