Journal: Science Advances

Article Title: Integrin inhibition facilitates fibrocartilaginous transformation in connective tissue in osteoarthritis

doi: 10.1126/sciadv.ady4112

Figure Lengend Snippet: ( A ) Pentachrome staining shows ECM changes in mouse ADD TMJ-OA disk tissues after cilengitide injection. Black dotted lines: boundary of the disk tissues. N = 3. Scale bars, 250 μm. ( B ) Semiquantification of mucin (bright blue) and collagen (yellow) expression via Movat pentachrome staining. Data are presented as mean ± SD. The two-tailed t test is used for data analysis. N = 3. #: not significant, * P < 0.05 versus normal group, and Ф: P < 0.05. ( C ) Semiquantification of Osteoarthritis Research Society International scores. Refer to references for grading criteria and method. Data are presented as mean ± SD. The two-tailed t test is used for data analysis. N = 6. * P < 0.05 and **** P < 0.0001. ( D ) FISH of ADD TMJ-OA disk tissues after cilengitide injection. Green: Col5a1 ; magenta: Has1 ; gray: DAPI. Yellow dotted lines: boundary of the disk tissues. White dotted lines: boundary of the glenoid fossa and condyle. Scale bars, 200 μm. ( E ) Semiquantification of Col5a1 + cell percentage and Col5a1 + / Has1 + cell percentage. Data are presented as mean ± SD. N = 3. The two-tailed t test is used for data analysis. ** P < 0.01 and n.s., not significant. ( F ) The heatmap of mechanical properties of mouse TMJ disk tissues via nanoindentation mapping. Data are normalized by the base-10 logarithm. ( G ) The average elastic modulus change of mouse TMJ disk tissues after cilengitide injection ( N = 4; data are presented as mean ± SD). * P < 0.05 and ** P < 0.01.

Article Snippet: MH7A cell line (BFN60805933, Bluefbio, China) was cultured in six-well plates and treated with 1 μM cilengitide (EMD 121974, MedChem Express, USA) for 48 hours.

Techniques: Staining, Injection, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: Integrin inhibition facilitates fibrocartilaginous transformation in connective tissue in osteoarthritis

doi: 10.1126/sciadv.ady4112

Figure Lengend Snippet: ( A ) Experimental design for scRNA-seq analysis of cilengitide-treated OA TMJ disk tissues. ( B ) UMAP plot of combined single-cell transcriptomic data from ADD TMJ-OA RCT with or without cilengitide injection ( N = 21,641). Cells are classified into six principal cell types. Green: Col5a1 high mFBs, N = 8210; light green: Col5a1 low mFBs, N = 8120; earthy yellow: mTLs, N = 371; blue: mECs, N = 1061; red: mMUs, N = 711; brown: mNEUs, N = 406; gray: mMACs, N = 2762. ( C ) Violin plot of Col5a1 expression in each mFBs. ( D ) Proportion changes of mFBs in RCT after cilengitide injection. Statistical tests are referred to Materials and Methods. **** P < 0.0001. ( E ) Activation of signaling pathways in mouse ADD TMJ-OA RCT after cilengitide injection. Each dot represents a signaling pathway, and the y axis indicates the activation level of the pathway. Values above 0.5 indicate increased pathway activation in ADD TMJ-OA with cilengitide RCT compared to ADD TMJ-OA RCT, while values below 0.5 show decreased activation. TGF-β signaling pathway is marked in red, and others are marked in green. ( F ) Immunohistochemical staining shows changes of TGF-β ligand expression in the retrodiscal tissues in ADD mouse with cilengitide injection. Black dotted lines: boundary of the disk tissues. N = 3. Scale bars, 250 μm. ( G ) Semiquantification of TGF-β expression via immunohistochemical staining. Data are presented as mean ± SD. N = 5 independent animals at each experimental condition. * P < 0.05 and ** P < 0.01. ( H ) Western blot of TGF-β signaling pathways and semiquantification of TGF-β expressions in mouse ADD RCT. Data are presented as mean ± SD. N = 5. * P < 0.05 and ** P < 0.01.

Article Snippet: MH7A cell line (BFN60805933, Bluefbio, China) was cultured in six-well plates and treated with 1 μM cilengitide (EMD 121974, MedChem Express, USA) for 48 hours.

Techniques: Injection, Expressing, Activation Assay, Protein-Protein interactions, Immunohistochemical staining, Staining, Western Blot

Journal: Science Advances

Article Title: Integrin inhibition facilitates fibrocartilaginous transformation in connective tissue in osteoarthritis

doi: 10.1126/sciadv.ady4112

Figure Lengend Snippet: ( A ) Schematic diagram of ADD TMJ-OA miniature pig model generation and strategy of cilengitide injection. ( B ) The macroscopic of TMJ disk tissues of miniature pigs in normal, ADD TMJ-OA, and ADD TMJ-OA + cilengitide group. Black: AD; yellow: retrodiscal tissue. Scale bars, 1 cm. ( C ) Pentachrome staining of TMJ disk tissues of miniature pigs in normal, ADD TMJ-OA, and ADD TMJ-OA + cilengitide group. Black: nuclei or elastic fibers; bright blue: mucin; bright red: fibrin. Scale bars, 500 μm. ( D ) The semiquantification of disk ECM changes of miniature pigs after ADD TMJ-OA and cilengitide treatment. ** P < 0.01 and **** P < 0.0001. ( E ) FISH of TMJ disk tissues of miniature pigs in normal, ADD TMJ-OA, and ADD TMJ-OA + cilengitide group. Green: Col5a1; gray: DAPI. Scale bars, 100 μm. ( F ) Semiquantification of Col5a1 + cell percentage. Data are presented as mean ± SD. N = 3 independent animals. The two-tailed t test was used for data analysis, * P < 0.01 and **** P < 0.0001. ( G ) Mach-1 mechanical analysis of TMJ disk tissues of miniature pigs in normal, ADD TMJ-OA, and ADD TMJ-OA + cilengitide group. The heatmap (top) shows the spatial distribution of the elastic modulus of disk tissues from different groups. The column graph (bottom) shows the average elastic modulus of the RCT from different group ( N = 3). The one-way analysis of variance (ANOVA) was used for data analysis. n.s., not significant and * P < 0.05. ( H ) The mechanism diagram of our scientific findings.

Article Snippet: MH7A cell line (BFN60805933, Bluefbio, China) was cultured in six-well plates and treated with 1 μM cilengitide (EMD 121974, MedChem Express, USA) for 48 hours.

Techniques: Injection, Staining, Two Tailed Test

Journal: Science Advances

Article Title: Dynamic adaptive coassembled sericin protein orchestrating stem cell development for nucleus pulposus regeneration

doi: 10.1126/sciadv.adx2768

Figure Lengend Snippet: ( A ) The composition and structure of the NP in IVD tissue exhibited spatial heterogeneity, which support NP cell development and physiological function through dynamic mechanical regulation and biochemical signaling. ( B ) Inspired by the partitioned structure of ECM, bioactive sericin was coassembled with BPAA-GFF fibrillar networks, driven by phase separation–induced molecular network rearrangement. The resulting dynamic adaptive network activated stem cell integrin ligands to modulate cell-matrix interactions and promoted chondrogenic differentiation via rapid stress relaxation and biochemical factor induction. ( C ) In rat and rabbit IVD degeneration models, KGN-incorporated coassembled hydrogel effectively promoted BMSCs for NP regeneration.

Article Snippet: To investigate the role of integrin β3 in mediating stem cell adhesion and cytoskeletal regulation, functional inhibition was performed using the αvβ3 integrin antagonist cilengitide (MedChemExpress, USA).

Techniques:

Journal: Science Advances

Article Title: Dynamic adaptive coassembled sericin protein orchestrating stem cell development for nucleus pulposus regeneration

doi: 10.1126/sciadv.adx2768

Figure Lengend Snippet: ( A ) 3D coculture of stem cells with the coassembled BS gel. ( B ) Time-dependent gene expression trend of stem cells regulated by coassembled BS protein gel. ( C ) Up-regulated genes and their associated biological events based on Gene Ontology (GO) analysis comparing BS versus B. ( D ) Flow relationships between activated molecular pathways and up-regulated genes based on Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis comparing BS versus B. JAK-STAT, Janus kinase–signal transducer and activator of transcription; MAPK, mitogen-activated protein kinase. ( E ) Gene interaction network analysis and Gantt chart analysis of temporal gene expression in three gene categories. ( F ) Gene expression heatmap analysis comparing BS versus B, n = 3 independent samples. ( G ) In vitro validation of gene expression by qPCR, n = 3 independent samples. ( H ) Local interaction residues in molecular docking. Color-coded representation shows BPAA-GFF (green), sericin protein (magenta), and the integrin αvβ3 complex (blue and orange). ( I ) Time-course analysis of stem cells cocultured with the hydrogel at 1, 2, and 4 weeks. ( J ) Integrin β3 expression analysis at 1 week, n = 3 independent samples. ( K ) Cytoskeletal staining and cell aspect ratio analysis at 1 week. ( L ) YAP staining and nuclear/cytoplasmic ratio analysis. ( M ) Coassembled BS gel activates integrin-mediated cell-matrix interactions, driving YAP nuclear translocation and downstream mechanotransduction signaling. Statistical analyses were performed with Student’s t tests (unpaired and two-tailed): * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: To investigate the role of integrin β3 in mediating stem cell adhesion and cytoskeletal regulation, functional inhibition was performed using the αvβ3 integrin antagonist cilengitide (MedChemExpress, USA).

Techniques: Gene Expression, In Vitro, Biomarker Discovery, Expressing, Staining, Translocation Assay, Two Tailed Test