Journal: Journal of Orthopaedic Translation

Article Title: Accumulation of advanced oxidation protein products aggravates bone-fat imbalance during skeletal aging

doi: 10.1016/j.jot.2024.12.010

Figure Lengend Snippet: AOPPs accumulation was accompanied by the bone-fat imbalance during skeletal aging. A Serum AOPPs levels in young mice (3-month-old) and aged mice (18-month-old) (n = 6). B AOPPs relative levels in bone marrow from young and aged mice (n = 6). C Serum ALP level (n = 6). D Serum FABP4 level (n = 6). E, F HE staining of bone trabeculae and adipocytes in distal femora, and the area of fat vacuoles was quantified. Scale bars = 100 μm. G, H IHC staining for OCN and number of osteoblasts in distal femoral trabeculas. Scale bars = 20 μm. I, J IHC staining for FABP4 and number of adipocytes. Scale bars = 20 μm. K, L Micro-CT scanning of osmium tetroxide-stained tibia and MAT content below the growth plate (n = 3). M, N The protein and mRNA expression of OCN and PPARγ in proximal tibial metaphysis. O Representative micro-CT images of distal femora from young and aged mice (n = 6) (Scale bars = 1 mm). P-V Quantitative trabecular and cortical bone parameters data were displayed in Bone mineral density (BMD), Trabecular Bone Volume/Total Volume (Tb.BV/TV), Trabecular Number (Tb.N), Trabecular thickness (Tb.Th), Trabecular Separation (Tb.Sp), Cortical Thickness (Ct.Th) and Cortical Bone Area (Ct.BArea) (n = 6). W-Y The correlation analysis of serum AOPPs and BMD, ALP, FABP4. Data were shown as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. young mice group (Student's t test).

Article Snippet: Next, the specimens were exposed overnight at 4 °C to osteocalcin (OCN) (sc-390877; Santa Cruz) and FABP4 (67167-1-Ig; Proteintech).

Techniques: Staining, Immunohistochemistry, Micro-CT, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: Accumulation of advanced oxidation protein products aggravates bone-fat imbalance during skeletal aging

doi: 10.1016/j.jot.2024.12.010

Figure Lengend Snippet: Decreased AOPPs level by antioxidant leaded to higher bone formation and lower marrow adiposity in aged mice. A Serum AOPPs levels in aged and NAC-treated aged mice (n = 6). B AOPPs relative levels in bone marrow from aged and NAC-treated aged mice (n = 6). C Serum ALP level (n = 6). D Serum FABP4 level (n = 6). E, F HE staining of bone trabeculae and adipocytes in distal femora, and the area of fat vacuoles was quantified. Scale bars = 100 μm. G, H IHC staining for OCN and number of osteoblasts in distal femoral trabeculas. Scale bars = 20 μm. I, J IHC staining for FABP4 and number of adipocytes. Scale bars = 20 μm. K, L Micro-CT scanning of osmium tetroxide-stained tibia and MAT content below the growth plate (n = 3). M, N The protein and mRNA expression of OCN and PPARγ in proximal tibial metaphysis. O Representative micro-CT images of distal femora from aged and NAC-treated aged mice (n = 6) (Scale bars = 1 mm). P-V Quantitative trabecular and cortical bone parameters data were displayed in BMD, Tb.BV/TV, Tb.N, Tb.Th, Tb.Sp, Ct.Th and Ct.BArea (n = 6). Data were shown as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. aged mice group (Student's t test).

Article Snippet: Next, the specimens were exposed overnight at 4 °C to osteocalcin (OCN) (sc-390877; Santa Cruz) and FABP4 (67167-1-Ig; Proteintech).

Techniques: Staining, Immunohistochemistry, Micro-CT, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: Accumulation of advanced oxidation protein products aggravates bone-fat imbalance during skeletal aging

doi: 10.1016/j.jot.2024.12.010

Figure Lengend Snippet: Chronic AOPPs loading lead to the bone-fat imbalance in young mice. A Serum AOPPs levels in 6-month-old mice and AOPPs-intervened mice (n = 6). B AOPPs relative levels in bone marrow from 6-month-old mice and AOPPs-intervened mice (n = 6). C Serum ALP level (n = 6). D Serum FABP4 level (n = 6). E, F HE staining of bone trabeculae and adipocytes in distal femora, and the area of fat vacuoles was quantified. Scale bars = 100 μm. G, H IHC staining for OCN and number of osteoblasts in distal femoral trabeculae. Scale bars = 20 μm. I, J IHC staining for FABP4 and number of adipocytes. Scale bars = 20 μm. K, L Micro-CT scanning of osmium tetroxide-stained tibia and MAT content below the growth plate (n = 3). M, N The protein and mRNA expression of OCN and PPARγ in proximal tibial metaphysis. O Representative micro-CT images of distal femora from AOPPs-intervened mice (n = 6) (Scale bars = 1 mm). P-V Quantitative trabecular and cortical bone parameters data were displayed in BMD, Tb.BV/TV, Tb.N, Tb.Th, Tb.Sp, Ct.Th and Ct.BArea (n = 6). Data were presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 vs. PBS group (one-way ANOVA).

Article Snippet: Next, the specimens were exposed overnight at 4 °C to osteocalcin (OCN) (sc-390877; Santa Cruz) and FABP4 (67167-1-Ig; Proteintech).

Techniques: Staining, Immunohistochemistry, Micro-CT, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: Accumulation of advanced oxidation protein products aggravates bone-fat imbalance during skeletal aging

doi: 10.1016/j.jot.2024.12.010

Figure Lengend Snippet: MSCs from aged mice tended to differentiate into adipocytes rather than osteoblasts and exhibited cellular senescence. A, B Representative images of Alizarin Red S (ARS) staining and quantitative analysis of matrix mineralization in MSCs from young and aged mice. C, D Representative images of Oil Red O (ORO) staining and quantitative analysis of lipid droplet formation in MSCs from young and aged mice. Scale bar = 100 μm. E, F Representative images of beta-galactosidase staining and quantitative analysis of senescent MSCs. Scale bar = 100 μm. G qRT-PCR analysis of the mRNA level of osteogenic markers (ALP, RUNX2 and OCN) 、adipogenic markers (PPARγ and C/EBPα) and senescent biomarkers (P53, P21 and P16) in MSCs from young and aged mice. H AOPPs relative levels in cellular supernatant of MSCs derived from young and aged mice. n = 3 each group. Data were shown as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. young mice group (Student's t test).

Article Snippet: Next, the specimens were exposed overnight at 4 °C to osteocalcin (OCN) (sc-390877; Santa Cruz) and FABP4 (67167-1-Ig; Proteintech).

Techniques: Staining, Quantitative RT-PCR, Derivative Assay

Journal: Journal of Orthopaedic Translation

Article Title: Accumulation of advanced oxidation protein products aggravates bone-fat imbalance during skeletal aging

doi: 10.1016/j.jot.2024.12.010

Figure Lengend Snippet: AOPPs induced the switch from osteogenic to adipogenic lineage and senescence in MSCs. A, B ARS staining and quantitative analysis of matrix mineralization in primary MSCs exposed to AOPPs (0–200 μg/ml). C Western blot analysis of the relative protein levels of ALP, RUNX2 and OCN in AOPPs-treated MSCs. D, E ORO staining and quantitative analysis of lipid droplets in primary MSCs exposed to AOPPs (0–200 μg/ml). Scale bar = 100 μm. F Immunoblotting analysis of PPARγ and C/EBPα protein levels in AOPPs-treated MSCs. G qRT-PCR analysis of the relative mRNA levels of ALP, RUNX2, OCN, PPARγ and C/EBPα. H, I Representative images of beta-galactosidase staining and quantitative analysis of senescent MSCs. Scale bar = 100 μm. J-M Protein levels of cellular senescence markers (P53, P21, P16), senescence-associated secretory phenotype (SASP) (MMP3, IL-6, IL-1β), senescence-associated heterochromatin foci (SAHF) (H3K9Me2), DNA damage (γ-H2AX) and multi-differentiation potential associated transcription factors (OCT-4, NANOG) were determined using immunoblotting. n = 3 each group. Data were presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 vs. control group (one-way ANOVA).

Article Snippet: Next, the specimens were exposed overnight at 4 °C to osteocalcin (OCN) (sc-390877; Santa Cruz) and FABP4 (67167-1-Ig; Proteintech).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Control

Journal: Journal of Orthopaedic Translation

Article Title: Accumulation of advanced oxidation protein products aggravates bone-fat imbalance during skeletal aging

doi: 10.1016/j.jot.2024.12.010

Figure Lengend Snippet: ROS signaling was involved in AOPPs-induced lineage switch and senescence of MSCs. A, B ARS staining and quantitative analysis of matrix mineralization in NAC-pretreated MSCs exposed to AOPPs. C Protein levels of ALP, RUNX2 and OCN induced by exposure of NAC pretreated MSCs to AOPPs. D, E ORO staining and quantitative analysis of lipid droplets. Scale bar = 100 μm. F Protein levels of PPARγ and C/EBPα. G mRNA levels of ALP, RUNX2, OCN, PPARγ and C/EBPα induced by exposure of NAC pretreated MSCs to AOPPs. H, I Representative images of beta-galactosidase staining and quantitative analysis of senescent MSCs. J-L Western blot analysis of the relative levels of P53, P21, P16, MMP3, IL-6, IL-1β, H3K9Me2 and γ-H2AX protein expression in NAC-pretreated MSCs exposed to AOPPs. n = 3 each group. Data were presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01 vs. control group. #p < 0.05 vs. AOPPs group (one-way ANOVA).

Article Snippet: Next, the specimens were exposed overnight at 4 °C to osteocalcin (OCN) (sc-390877; Santa Cruz) and FABP4 (67167-1-Ig; Proteintech).

Techniques: Staining, Western Blot, Expressing, Control

Journal:

Article Title: Therapeutic effect of intracolonically administered nuclear factor ? B (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis

doi: 10.1046/j.1365-2249.2000.01183.x

Figure Lengend Snippet: Immunohistochemical study of the rectal tissue in mice with DSS-induced colitis. Rectal tissue was stained with mouse MoAb F4/80 on (a) day 0, (b) day 3, and (c) day 7 and inspected with a microscope (× 400). F4/80+ macrophages were observed at the base of crypts on day 3. On day 7, considerable numbers of macrophages were observed in the entire field of the lamina propria. In each section, some spindle-shaped cells, presumably fibroblasts, were stained. However, on day 7 the numbers of spindle-shaped cells stained by F4/80 MoAb were not increased compared with those of macrophages, polymorphic shaped cells stained its MoAb.

Article Snippet: Mice were killed on days 0, 3 or 7, and rectal specimens were stained with anti-mouse macrophage MoAb, F4/80 (Cedarlane, Hornby, Ontario, Canada).

Techniques: Immunohistochemical staining, Staining, Microscopy

Journal: Materials

Article Title: Evaluation of Preosteoblast MC3T3-E1 Cells Cultured on a Microporous Titanium Membrane Fabricated Using a Precise Mechanical Punching Process

doi: 10.3390/ma13225288

Figure Lengend Snippet: Relative gene expression of OCN (a) and OPN (b) on day 14 and 28. MC3T3-E1 cells on specimens were cultured in basal and osteogenic medium (OS). Real-time PCR data were calculated from independent samples ( n ≥ 3). All values are shown as the mean ± standard deviation ( n ≥ 3). All results were compared by a one-way ANOVA with Tukey’s test using SPSS Statistics 17. The results with p values < 0.05 were deemed to be significant (* p < 0.05, ** p < 0.01, *** p < 0.001); n.s., not significant ( p > 0.05).

Article Snippet: Specimens were incubated with a rat anti-osteocalcin (OCN) antibody (1:200 dilution, Takara Bio Inc., Kusatsu, Japan) and rabbit anti-osteopontin (OPN) antibody (1:1000 dilution, Abcam, Cambridge, MA, USA) for 1 h, followed by an incubation with an Alexa Fluor ® 488-conjugated goat anti-mouse secondary antibody (1:1000 dilution, Abcam) and Alexa Fluor ® 555-conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Abcam) for 1 h. Specimens were also incubated with 4′,6-diamidino-2-phenylinole (DAPI) (Invitrogen TM /Life Technologies) diluted in PBS for 5 min to identify nuclei.

Techniques: Gene Expression, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Materials

Article Title: Evaluation of Preosteoblast MC3T3-E1 Cells Cultured on a Microporous Titanium Membrane Fabricated Using a Precise Mechanical Punching Process

doi: 10.3390/ma13225288

Figure Lengend Snippet: Immunofluorescence images of Ti surface-related OCN and OPN expression in MC3T3-E1 cells. Cells were incubated in α-MEM (5% FBS) with an osteogenic supplement for 7, 14 and 21 days. Nuclei, OCN and OPN were visualized by staining with DAPI (blue), Alexa Fluor ® 488 (green) and Alexa Fluor ® 555 (red). Scale bars represent 100 μm. ( a – c ) were for 7, 14, and 21 days respectively.

Article Snippet: Specimens were incubated with a rat anti-osteocalcin (OCN) antibody (1:200 dilution, Takara Bio Inc., Kusatsu, Japan) and rabbit anti-osteopontin (OPN) antibody (1:1000 dilution, Abcam, Cambridge, MA, USA) for 1 h, followed by an incubation with an Alexa Fluor ® 488-conjugated goat anti-mouse secondary antibody (1:1000 dilution, Abcam) and Alexa Fluor ® 555-conjugated goat anti-rabbit secondary antibody (1:1000 dilution, Abcam) for 1 h. Specimens were also incubated with 4′,6-diamidino-2-phenylinole (DAPI) (Invitrogen TM /Life Technologies) diluted in PBS for 5 min to identify nuclei.

Techniques: Immunofluorescence, Expressing, Incubation, Staining

Journal: Theranostics

Article Title: Macrophage targeted theranostic strategy for accurate detection and rapid stabilization of the inflamed high-risk plaque

doi: 10.7150/thno.59759

Figure Lengend Snippet: In vitro and in vivo feasibility study of MMR-Lobe-Cy. (A-C) Expression of CD206 in foam cells. (A) CD206 ( Mrc1 ) mRNA expression in non-stimulated control macrophages and foam cells by qPCR analysis. * P < 0.05 by Mann-Whitney U test. (B) Representative immunofluorescence images of non-stimulated control cells (left) and foam cells (right) labeled with CD206 antibody (green). Scale bar, 50 µm. (C) Flow cytometry analysis of CD206 expression in control (left) and foam cells (right). Red colored area indicates negative control (staining only with secondary antibody). (D) CLSFM images of MMR-Lobe-Cy internalization in foam cells (MMR-derived NIRF in red). The cellular uptake of MMR-Lobe-Cy decreased when mannose receptors on foam cells were blocked with pre-treated free mannan. Scale bars, 30 µm. * P < 0.05 by Kruskal-Wallis test followed by Mann-Whitney U test with Bonferroni correction. (E) In vitro anti-inflammatory effects of MMR-Lobe-Cy on foam cells as assessed by the expression of inflammatory mediators including MCP-1, MMP-9, IL-1β, and IL-6 by ELISA. Mannose receptor blocking assay with mannan showed the neutralized effects of MMR-lobe-Cy from the foam cells. * P < 0.05 by one-way ANOVA followed by Tukey's post-hoc test. (F) Representative ex vivo fluorescence imaging of the harvested liver, kidney, spleen, and lung of MMR-Lobe-Cy after 24 h post-injection in rabbits (left), and quantitative fluorescence analysis of tissue biodistribution (n = 6) (right). Data represent the means ± standard error of the mean. * P < 0.05 by Kruskal-Wallis test followed by Mann-Whitney U test with Bonferroni correction. (G) Time-dependent blood pharmacokinetics of MMR-Lobe-Cy in a rabbit. After collecting blood sample at predetermined time intervals, NIRF intensity of MMR-Lobe-Cy at each tme step was measured and fitted to calculate the estimated blood half-life time. (H) Representative result of in vivo intravascular longitudinal OCT-NIRF imaging, in vivo 2-dimensional (2D) NIRF mapping, and ex vivo FRI. The corresponding in vivo axial OCT-NIRF cross-sections, FM images (MMR-derived NIRF in red, autofluorescence in green), and RAM11 immunostained tissue images were designated by white dotted lines; (a) non-injured (normal) and (b) balloon-injured (plaque) arterial segments. Scale bar, 500 µm. Equally windowed.

Article Snippet: To examine macrophages and protease expression, the specimens were stained with RAM11 anti-macrophage (1:1000 dilution) and 4A3 anti-MMP-9 (1:100 dilution; Novus Biologicals).

Techniques: In Vitro, In Vivo, Expressing, MANN-WHITNEY, Immunofluorescence, Labeling, Flow Cytometry, Negative Control, Staining, Derivative Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Ex Vivo, Fluorescence, Imaging, Injection

Journal: Theranostics

Article Title: Macrophage targeted theranostic strategy for accurate detection and rapid stabilization of the inflamed high-risk plaque

doi: 10.7150/thno.59759

Figure Lengend Snippet: Ex vivo FRI NIRF imaging for confirmation of in vivo findings. (A) Comparison of representative in vivo NIRF 2-dimensional (2D) mapping between Day 0 (1st row) vs. Day 7 (2nd row), and pTBR/Frame (3rd row) along the pullback direction. NIRF signals decreased after MMR-Lobe-Cy treatment at Day 7 (1st column), whereas the signals tended to increase after administration with saline (3rd column). Prox., proximal; Dist., distal. Scale bars, 4 mm. Equally windowed. (B) Ex vivo FRI NIRF (top), brightfield (middle), and merged images (bottom) of resected arteries. Scale bars, 4 mm. Equally windowed. (C) Pearson correlation analysis of in vivo and ex vivo mean pTBR. (D) A linear relationship between in vivo vs. ex vivo pTBR/Frame values of the corresponding sites of the artery ( r : Spearman correlation coefficient). (E) Quantitative comparison of ex vivo mean pTBR comparison among each group. * P < 0.05 by Kruskal-Wallis test followed by Mann-Whitney U test with Bonferroni correction. ns, non-significant. (F-H) Co -relationship between in vivo axial OCT-NIRF images and histological validation. (F) The representative in vivo axial OCT-NIRF cross-sectional images of each group correspond with the FM images (MMR-derived NIRF in red, autofluorescence in green), macrophage immunostaining (RAM11), haematoxylin and eosin staining (H&E), and oil red O (ORO) lipid staining, respectively. Scale bars, 500 µm. Equally windowed. (G) Quantitative analysis of RAM11-positive macrophage areas of each treatment group including MMR-Lobe-Cy, oral lobeglitazone, and saline control. (H) ORO staining area comparison for lipid composition of each group. ** P < 0.01, *** P < 0.001 by Kruskal-Wallis test followed by Mann-Whitney U test with Bonferroni correction. ns, non-significant.

Article Snippet: To examine macrophages and protease expression, the specimens were stained with RAM11 anti-macrophage (1:1000 dilution) and 4A3 anti-MMP-9 (1:100 dilution; Novus Biologicals).

Techniques: Ex Vivo, Imaging, In Vivo, Comparison, Saline, MANN-WHITNEY, Derivative Assay, Immunostaining, Staining

Journal: Theranostics

Article Title: Macrophage targeted theranostic strategy for accurate detection and rapid stabilization of the inflamed high-risk plaque

doi: 10.7150/thno.59759

Figure Lengend Snippet: In vivo evaluation of macrophage contents, protease expression, and collagen contents in rabbit atheroma after 14 days of MMR-Lobe-Cy treatment. Comparison of (A) RAM11 macrophage contents and (B) MMP-9 expression in rabbit atheroma. PSR-stained plaques imaged with (C) brightfield microscopy to show total collagen contents and (D) polarized microscopy to evaluate collagen type I contents. n = 3 segments per rabbit, n = 3 rabbits per group. Scale bar, 200 µm. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Kruskal-Wallis test followed by Mann-Whitney U test with Bonferroni correction.

Article Snippet: To examine macrophages and protease expression, the specimens were stained with RAM11 anti-macrophage (1:1000 dilution) and 4A3 anti-MMP-9 (1:100 dilution; Novus Biologicals).

Techniques: In Vivo, Expressing, Comparison, Staining, Microscopy, MANN-WHITNEY