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: Continuous intraosseous administration of SCS prevents glucocorticoid-induced bone degeneration. ( A ) Schematic illustration of the glucocorticoid (GC; MPS)-induced bone deterioration and intraosseous SCS treatment. ( B-D ) Representative H&E staining images of the femur at 6 weeks (B). Magnified views of the cortical bone and trabecular bone in the marrow cavity are shown on the right. Solid arrows indicate normal osteocytes, while hollow arrows indicate empty osteocyte lacunae. Quantification of empty lacunae ratios in cortical bone (C) and trabecular bone (D). n = 6 biological replicates. (Scale bars, 500 μm and 25 μm) ( E-H ) Representative immunofluorescence staining of OPN + mature osteoblasts, osteolectin + osteoprogenitors, and VE-cadherin + endothelial cells (ECs) in femur at 6 weeks (E), and corresponding quantifications (F–H). n = 6 biological replicates. (Scale bars, 100 μm and 20 μm) ( I and J ) Representative flow cytometry plots of capillary subtypes in the femur (I), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (J). n = 6 biological replicates. ( K and L ) Flow cytometry plots showing Sca-1 hi CD31 hi arteriolar ECs (K), and corresponding quantification (L). n = 6 biological replicates. ( M and N ) Representative micro-CT 3D images of the femur (M). Quantitative analysis of percent bone volume (BV/TV) (N). n = 6 biological replicates. (Scale bars, 1.5 mm, 600 μm and 545 μm) ( O and P ) ELISA analysis of VEGF (O) and PDGF-BB (P) levels in bone marrow supernatant and peripheral serum from PBS- and SCS-treated groups at week 6. n = 6 biological replicates. ( Q ) ELISA quantification of the osteogenic factor osteocalcin in peripheral serum at week 6. n = 6 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 one-way ANOVA with Tukey's post hoc test ( C, D, F, G, H, J, L, N, O, P and Q ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Staining, Immunofluorescence, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay

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 attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

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: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

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

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 reprograms the lineage commitment of MSCs after GC treatment and inhibits the generation of primary senescent adipocytes. ( A ) Schematic illustration of the in vitro investigation of SCS targeting the prostaglandin/PPARγ/INK positive feedback loop in MPS-induced primary senescent adipocytes. ( B ) Representative flow cytometry plot showing p16 + senescent cells in adipocytes derived from bone marrow after 14 days of in vivo MPS induction and subsequently treated with SCS in vitro . ( C ) qPCR analysis of 12 senescence-associated markers in primary senescent adipocytes after in vitro SCS treatment. n = 3 biological replicates. ( D ) ELISA analysis of IL-1β levels in adipocyte supernatant following in vitro SCS treatment. n = 6 biological replicates. ( E ) ELISA analysis of secreted prostaglandins PGD2 and PGE2 in adipocytes under different treatment conditions. D-PBS: bone marrow adipocytes isolated from mice treated in vivo with the solvent control DMSO, followed by in vitro treatment with PBS; M-PBS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with PBS. M-SCS: bone marrow adipocytes isolated from mice treated in vivo with MPS, followed by in vitro treatment with SCS. ( F ) Western blot analysis of intracellular COX-2 protein levels in adipocytes across the three treatment conditions. ( G ) Schematic illustration of competitive osteogenic–adipogenic differentiation of CD45 − Ter119 − CD31 − LepR + MSCs after 7 days of in vivo SCS and MPS co-treatment. ( H ) qPCR analysis of pan-adipocyte markers ( Fabp4 , Adipoq , Plin1 , Cd36 , and Lep ) in CD45 − Ter119 − CD31 − LepR + MSCs after 14 days of in vitro competitive lineage differentiation. n = 3 biological replicates. ( I and J ) Representative immunofluorescence images (I) and quantification (J) of perilipin + adipocytes and osteopontin + mature osteoblasts derived from lineage-committed MSCs. n = 6 biological replicates. (Scale bars, 30 μm, 15 μm and 15 μm). ( K ) Western blot analysis of adipogenesis-related markers C/EBPα, PPARγ, and C/EBPβ in the lineage-mixed cells after in vitro competitive differentiation of CD45 − Ter119 − CD31 − LepR + MSCs. ( L ) qPCR analysis of lipogenesis-related markers Fasn , Scd1 , Srebf1 , Acaca , and Acacb . n = 3 biological replicates. ( M and N ) Representative H&E staining images (M) of the femurs at day 14 following SCS and MPS co-treatment. Yellow arrows indicate bone marrow adipocytes. Magnified images show hypertrophic adipocyte morphology, with quantification of adipocyte diameter (N). n = 19 biological replicates. (Scale bars, 200 μm, 50 μm and 20 μm). 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 ( C, D, H, J, L and N ), or one-way ANOVA with Tukey's post hoc test ( E ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: In Vitro, Flow Cytometry, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Solvent, Control, Western Blot, Immunofluorescence, Staining, Two Tailed Test

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: Gene expression profiles of bone marrow-derived LepR + MSCs after 7-day in vivo co-treatment with SCS and MPS. ( A ) Heatmap showing DEGs in CD45 − Ter119 − CD31 − LepR + MSCs sorted from bone marrow at day 7 post-treatment with SCS versus PBS ( P < 0.05, |log fold change| > 1.5). n = 3 biological replicates. ( B ) Representative GO biological process enrichment analysis of downregulated DEGs. ( C ) Top 20 enriched KEGG pathways of downregulated DEGs in SCS versus PBS. ( D ) GSEA plots of biological processes positively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25). ( E ) Representative downregulated DEGs associated with adipogenesis and lipogenesis identified through KEGG pathway analysis. n = 3 biological replicates. ( F ) Top 20 enriched KEGG pathways of upregulated DEGs in SCS versus PBS. ( G ) Representative GO biological process enrichment analysis of upregulated DEGs. ( H ) Representative upregulated DEGs identified through biological process enrichment analysis. n = 3 biological replicates. ( I and J ) GSEA plots of KEGG pathways negatively enriched in the SCS group (|NES| > 1, nominal P < 0.05, FDR <0.25).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Gene Expression, Derivative Assay, In Vivo

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 targets downstream senescent lineage commitment of bone marrow MSCs to mitigate GC-induced bone deterioration. ( A ) Schematic diagram illustrating the experimental design: CD45 − Ter119 − CD31 − LepR + MSCs isolated from mice co-treated with SCS and MPS for 7 days were subjected to in vitro lineage-competitive differentiation, followed by DEX-induced senescence in lineage-mixed cells. These cells were then adoptively transplanted into healthy bone marrow cavity to assess bone deterioration development. ( B ) Representative H&E-stained images of the femur 12 weeks after adoptive transfer. PBS-DEX group: LepR + MSCs from PBS and MPS co-treated mice subjected to in vitro lineage differentiation and DEX-induced senescence, followed by transplantation. SCS-DEX group: LepR + MSCs from SCS and MPS co-treated mice processed similarly. PBS group: solvent control without cell transplantation. Solid arrows indicate intact osteocytes; hollow arrows indicate empty lacunae. (Scale bars, 250 μm and 25 μm) ( C – E ) Quantitative analysis of marrow hypertrophic adipocyte diameter (C), proportion of empty osteocyte lacunae in trabecular bone (D), and adipocyte number (E) in the metaphysis 12 weeks post-transplantation. n = 19 biological replicates (C), n = 6 biological replicates (D), n = 8 biological replicates (E). ( F ) Quantification of empty lacunae in epiphysis at 12 weeks post-transplantation. n = 6 biological replicates. ( G – I ) Representative flow cytometry plots of capillary ECs subtypes in the femur at 12 weeks (G), with quantification of CD45 − Ter119 − CD31 hi Emcn hi ECs (H) and CD45 − Ter119 − CD31 lo Emcn lo ECs (I). n = 6 biological replicates. ( J and K ) Representative flow cytometry plots (J) and corresponding quantification (K) of CD45 − Ter119 − Sca-1 hi CD31 hi arteriolar ECs in the femur at 12 weeks post-transplantation. n = 6 biological replicates. ( L ) Representative micro-CT images of the femur at 12 weeks post-transplantation across different treatment groups. (Scale bars, 1.5 mm and 500 μm) ( M – P ) Quantitative analysis of bone parameters in the metaphysis: bone mineral density (BMD) (M), percent bone volume (BV/TV) (N), trabecular separation (Tb.Sp) (O), and trabecular number (Tb.N) (P). n = 6 biological replicates. ( Q ) Serum ELISA analysis of the osteogenic marker osteocalcin at 12 weeks post-transplantation. n = 6 biological replicates. ( R and S ) ELISA analysis of PDGF-BB (R) and VEGF (S) in both bone marrow supernatant and peripheral serum at 12 weeks post-transplantation. n = 6 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 one-way ANOVA with Tukey's post hoc test ( C, D, E, F, H, I, K, M, N, O, P, Q, R and S ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Isolation, In Vitro, Staining, Adoptive Transfer Assay, Transplantation Assay, Solvent, Control, Flow Cytometry, Micro-CT, Enzyme-linked Immunosorbent Assay, Marker

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 modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. 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 ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: Following washing with buffer, cells were incubated with APC streptavidin at 4 °C for 40 min. After washing, CD45 − Ter119 − CD31 − LepR + MSCs were sorted using the MACSQuant® Tyto® cell sorter (Miltenyi Biotec).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: Journal of Translational Autoimmunity

Article Title: T-cell immunity in the experimental autoimmune vasculitis rat model

doi: 10.1016/j.jtauto.2025.100305

Figure Lengend Snippet: Dynamics of T-cell immunity in the experimental autoimmune vasculitis model. (A) mRNA analysis of the kidney by RT-PCR revealed a persistent rise of IL-17A transcripts in MPO-immunized animals over six weeks. In contrast, IFNγtranscripts remained stable with no clear rise over time. The horizontal line depicts the median. The fold change over control was calculated using the Δ Δ CT method, rats receiving Freund's adjuvant without MPO served as controls. (B) Th17 and Th1 cells were present in inflamed kidneys as early as two weeks after immunization with MPO. The fraction of Th1 cells remained on the same level whereas the proportion of Th17 cells sharply increased over time and peaked at week six. T-cells were analyzed by FACS. (C) Representative flow cytometric data of renal Th17 cells. The plots are gated on CD45 + CD3 + T-cells. Data is shown as mean with SD- (D) MPO-specific renal Th17 and Th1 cells were detectable in MPO-immunized animals but not in controls. Elispot was used to detect antigenspecific T-cells. The horizontal line depicts the median. Mann-Whitney U Test was used to calculate statistical significances. A p-value below 0.05 was considered significant. ∗p-value<0.05 ∗∗p-value <0.005.

Article Snippet: The single cell suspension from renal tissue was incubated with anti-rat CD45 (PE labelled, Miltenyi Biotec, Mönchengladbach, Germany) for 30 min at 4 °C followed by two washing steps with PBS containing 0.5 % BSA and 2 mM EDTA.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Adjuvant, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Journal of the American Chemical Society

Article Title: Glucose-Powered Ultrasmall Chemotactic Nanorobots for Retinal Degeneration Treatment

doi: 10.1021/jacs.5c15651

Figure Lengend Snippet: In vivo tests of nanorobots’ effect on the retina in the healthy mouse model. Positive cell percentage with NPs when intravitreally injected with two different dosages and compared with nontreated control and PBS-injected control: (a) retina after 1-day treatment; (b) posterior segment after 1-day treatment; (c) lymph nodes after 1-day treatment; (d) retina after 7-day treatment; (e) posterior segment after 7-day treatment; and (f) lymph nodes after 7-day treatment. (g) Schematic illustration of intravitreal injection into mouse eyes toward the retina (vertical cross-section of the eye and retina). The scheme is created with BioRender.com . (h) Positive rhodopsin cell percentage in the retina after 1 day of NPs intravitreal treatment. (i) CD45 + cell percentage in the retina after 1 day of NPs intravitreal treatment. (j) Positive rhodopsin cell percentage in the retina after 7 days of NPs intravitreal treatment. (k) CD45 + cell percentage in the retina after 7 days of NPs intravitreal treatment. The number of tested mice in each group is n ≥ 6. Data were presented as mean ± s.e.m. P values were analyzed by a two-sample t test, where NS represents nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The CD11b + CD45 + population was separated using a MidiMACS separator with attached LS Columns (both Miltenyi Biotec).

Techniques: In Vivo, Injection, Control

Journal: Journal of the American Chemical Society

Article Title: Glucose-Powered Ultrasmall Chemotactic Nanorobots for Retinal Degeneration Treatment

doi: 10.1021/jacs.5c15651

Figure Lengend Snippet: Effect of in vivo application of NPs into the degenerated retina. (a) In vivo acute retinal degeneration was established by intraperitoneal injection of NaIO 3 into healthy mice after 10 days for acclimation (counted as day 0) to induce selective damage of RPE cells and photoreceptors (rhodopsin-positive cells). On day 1 after intravitreal injection, tested NPs were intravitreally applied into the eye with degenerated retina. On day 8 (7 days after NPs applications), mice were sacrificed, and retinal samples were harvested for further analysis. (b) Representative density dot plots of infiltration of the degenerated retina with CD11b + cells and CD11b + CX3CR1 + cells (microglia/macrophages). (c) Infiltrations of degenerated retinas with different types of NPs. (d) Number of NPs + CD11b + double-positive cells in the degenerated retinas. (e) Expression of genes for rhodopsin , Iba-1 , Gfap, IL-1 β, superoxide dismutase-1 , catalase , Galectine-3 , and Bax were assessed by real-time PCR in the healthy retina (−), degenerated untreated retina (+), and degenerated retina (+) treated with CeNPs, Au 1 –CeNPs, and TPP-Au 11 –CeNPs. The number of mice in each group is n ≥ 10. Data were presented as mean ± s.e.m. P values were analyzed by a two-sample t test, where NS, nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The CD11b + CD45 + population was separated using a MidiMACS separator with attached LS Columns (both Miltenyi Biotec).

Techniques: In Vivo, Injection, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of the American Chemical Society

Article Title: Glucose-Powered Ultrasmall Chemotactic Nanorobots for Retinal Degeneration Treatment

doi: 10.1021/jacs.5c15651

Figure Lengend Snippet: Effect of in vitro nanorobot-immune cell regulation mechanism. (a) Schematic illustration of the purification and isolation of murine-bone marrow derived CD11b + CD45 + cells. The scheme is created with BioRender.com . (b) Cell viability of CD11b + CD45 + cells incubated with three types of NPs at a concentration of 125 pg NP/cell after 48 h. (c) Representative dot plots of infiltration of the DiI tagged NPs to the CD11b + CD45 + cells at different time points at 10 min, 1 h, and 24 h. (d) Expression of genes for IL-1 β, TNF- α, and VEGF in the NP-treated CD11 + CD45 + cells. (e) Protein production of IL-1β and MHC II, and the measured NO production in the NP-treated CD11 + CD45 + cells. The number in each group is n ≥ 3. Data were presented as mean ± s.e.m. P values were analyzed by a two-sample t test, where NS, nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The CD11b + CD45 + population was separated using a MidiMACS separator with attached LS Columns (both Miltenyi Biotec).

Techniques: In Vitro, Purification, Isolation, Derivative Assay, Incubation, Concentration Assay, Expressing