Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: In addition, ELISA analyses of senescence-associated inflammatory factors (IL-1β, IL-18, TNF-α, CCL3 (Neobioscience, EMC010a.96), CXCL1 (R&D Systems, MKC00B-1), and IL-6 (Neobioscience, EMC004.96)) were conducted on day 28 following combined treatment with SCS and rapamycin and/or T0070907.

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test