Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Schematic Illustration of Preparation of IL4-Loaded NHG-MSa

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques:

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Characterizations of IL4-loaded NHG-MS and NG-MS and their release profiles. (A–C) Stacked confocal images of IL4-loaded NHG-MS. (D–F) Cross-sectional confocal images at a higher magnification, showing that IL4 (red) was evenly distributed in the NHG-MS (green). (G) Loading efficiency of IL4 in NHG-MS and NG-MS. (H) Release profiles of IL4 from NHG-MS and NG-MS. (I) Total amount of IL4 (released and unreleased) from the NHG-MS and NG-MS detected via an ELISA (**P < 0.01).

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: In vitro bioactivity assay of the IL4 released from NHG-MS. Bone marrow-derived macrophages (BMDMs) stimulated by LPS and IFN-γ for 24 h were polarized into M1 proinflammatory macrophages and then exposed to IL4 released from NHG-MS at 1 and 14 days for 24 h. The LPS + IFN-γ-only and the LPS + IFN-γ + IL4 groups were used as negative and positive controls, respectively. (A, B) Immunofluorescence images and quantitative analysis of CD206+ (green, an M2 macrophage marker) and CD68+ (red, pan-macrophage marker) BMDMs. Cells treated with the LPS + IFN-γ-only group exhibited a rounded morphology, and CD206+ cells were barely detected in this negative control group, indicating the proinflammatory M1 phenotype. By contrast, addition of IL4 released from NHG-MS at 1 and 14 days switched the cells into typical elongated spindle shapes, and approximately 70% BMDMs were CD206+CD68+ M2 phenotype, which was similar to the IL4 positive control group. (C, D) Quantitative analysis of proinflammatory cytokines in the supernatant showed the significant reduction of both TNF-α and IL6 in three groups with additional IL4 treatment (**P < 0.01).

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: In Vitro, Derivative Assay, Immunofluorescence, Marker, Negative Control, Positive Control

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Macrophage phenotype in the defect area 7 days after surgical operation: iNOS (green, an M1 macrophage marker); CD206 (green, an M2 macrophage marker); and CD68 (red, pan-macrophage marker). (A) Immunofluorescence images showed that iNOS was extensively expressed in the DM and DM + NHG-MS groups. In comparison, the expression of iNOS in the IL4-loaded NHG-MS DM group was much less, which is similar to the normal control group, indicating fewer M1 macrophages in the normal control and IL4-loaded NHG-MS DM groups than the other two DM groups. (B) Immunofluorescence images showed that the expression of CD206 in the IL4-loaded NHG-MS DM group was similar to that in the normal control group and much higher than that in the other two DM groups. (C–F) Quantitative analysis showed that the expression of CD68 was significantly higher in the three DM groups than that in the normal control group (C), indicating that more macrophages infiltrated into the defect site of diabetic rats. Fewer M1 macrophages and more M2 macrophages in both the normal control and IL4-loaded NHG-MS DM groups (D, E) resulted in significantly higher M2/M1 ratios than in the other two DM groups (F) (**P < 0.01).

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: Marker, Immunofluorescence, Comparison, Expressing, Control

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Expression of proinflammatory cytokines TNF-α in the defect area 7 days after the surgical operation. (A–D) Immunohistochemical images showed stronger expression of TNF-α in DM and DM + NHG-MS groups than in normal control and IL4-loaded NHG-MS DM groups. (E, F) Quantitative analysis of TNF-α positive area ratio and mean integral optical density (IOD) (**P < 0.01).

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: Expressing, Immunohistochemical staining, Control

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Expression of Osterix and Runx2 in the defect area 14 days after the surgical operation. (A, B) Immunohistochemical images showed significantly more Osterix+ and Runx2+ osteoprogenitor cells in normal control and IL4-loaded NHG-MS DM groups than in the other two DM groups. (C–F) Quantitative analysis of the number of Osterix+ and Runx2+ cells and mean integral optical density (IOD) (*P < 0.05, **P < 0.01).

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: Expressing, Immunohistochemical staining, Control

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: ALP staining of the defect area 14 days after the surgical operation. (A–D) Normal control and IL4-loaded NHG-MS DM groups exhibited significantly more extensive and robust ALP expression than in the other two DM groups. (E, F) Quantitative analysis of ALP-positive area ratio and mean integral optical density (IOD) (*P < 0.05, **P < 0.01).

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: Staining, Control, Expressing

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: μ-CT three-dimensional reconstruction images of the rat mandible and new regenerated bone 4 weeks after surgery. (A–H) The defect area was entirely occupied by new regenerated bone in both the normal control group and the IL4-loaded NHG-MS DM group. In contrast, only half of the defect region was filled with new bone in the DM and DM + NHG-MS groups. (I) Ratio of bone volume to total volume (BV/TV) in all four groups (**P < 0.01).

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: Control

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: HE staining of the defect area 4 weeks after the surgical operation. (A–D) Newly formed bone filled the entire defect area in the normal control and the IL4-loaded NHG-MS DM group. In comparison, new bone formation was observed on the outer boundary of the defect area in the DM and DM + NHG-MS groups. The yellow arrows indicate newly formed bone, and the blue arrows indicate microspheres.

Article Snippet: Next, the blocking solution was aspirated and the NHG-MS was incubated in antirat IL4 primary antibody solution (1:100 in 2% goat serum, Peprotech) at room temperature for 2 h. After washing with PBST (0.05% Tween-20 in PBS) three times, the NHG-MS was incubated in Alexa Fluor 647 (red) antirabbit IgG solution (1:200 in 2% goat serum, Abcam) at room temperature for 1 h. The samples were observed under a confocal laser scan microscope (TCS SP5, Leica, Buffalo).

Techniques: Staining, Control, Comparison

Journal: The FASEB Journal

Article Title: Circadian period 2: a missing beneficial factor in sickle cell disease by lowering pulmonary inflammation, iron overload, and mortality

doi: 10.1096/fj.201900246RR

Figure Lengend Snippet: Genetic deletion of Per1/Per2 enhances pulmonary leukocyte infiltration and lung tissue damage, which contribute to an early mortality in SCD. A) Schematic of SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. B) Images of irradiated male mice ∼16 wk post-BMT. C) Increased mortality was identified beginning 42 d and up to 112 d after irradiation and hematopoietic adoptive transfer in female SCD BM transplanted to Per1/Per2 dKO mice. D) Panel of histologic stains, hematoxylin and eosin (H&E), and Ly6-positive leukocytes as indicated with red arrows in SCD or WT BM transplanted to WT or Per1/Per2 dKO mice. E) Semiquantification of histologic assessment of lung isolated from SCD or WT phenotypic mice with or without Per1/Per2. F) Quantification of leukocytes in ×20 field in whole lung sections isolated from SCD and WT BM transplant mice; n = 5 mice/group. ND, not determined. *P < 0.05, SCD → WT compared with WT → WT mice, **P < 0.01, SCD → WT compared with SCD → Per1/Per2 dKO mice.

Article Snippet: Sections were incubated with Ly6 primary antibody (rat monoclonal anti-mouse, 1:500 dilution in blocking solution; BD Biosciences, San Jose, CA, USA) overnight at 4°C.

Techniques: Irradiation, Adoptive Transfer Assay, Isolation