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Results: 6

1.
Fig. 6

Fig. 6. From: Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification.

Immunohistochemical staining for brown adipocytes expressing VEGF-D (green, c) in tissues isolated from the Flk1-H2B::YFP mice 4 days after receiving MC3T3 cells transduced with Ad5-BMP-2. Brown adipocytes were identified as cells expressing uncoupling protein 1 (UCP 1; red, d) and yellow (b) represents the Flk-yfp+ endothelial cells within the muscle. The tissues also were stained with VEGF-D antibodies (c) and counterstained with DAPI (blue, a), which stains the nucleus of cells. A merger of these stains (UCP-1, VEGF-D, and YFP) is shown in panel e. In panel f, a paraffin section taken 4 days after injection of BMP-2-producing cells was stained with an antibody against UCP1, and staining was visualized using 3,3'-diaminobenzidine (DAB) as described previously.(8) No staining was observed on a paraffin section taken 4 days after injection of cells transduced with the empty control vector Ad5-HM4 (data not shown).

Christine Fouletier Dilling, et al. J Bone Miner Res. 2010 May;25(5):1147-1156.
2.
Fig. 1

Fig. 1. From: Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification.

Immunohistochemical analysis of endothelial cell replication in tissues isolated at daily intervals after induction of bone formation with cells expressing BMP-2. (A–E) On days 1, 2, 3, 4, and 5, respectively, after injection of BMP-2-producing cells, paraffin sections were prepared and stained with an antibody against Ki67, followed by a secondary antibody conjugated to Alexa fluor 488 (green) mixed with an anti–von Willibrand Factor (vWF) antibody, followed by a secondary antibody conjugated to Alexa fluor 547 (red). (F) A representative image, similar staining, taken from tissues isolated from mice injected with cells transduced with a control vector (Ad5-empty).

Christine Fouletier Dilling, et al. J Bone Miner Res. 2010 May;25(5):1147-1156.
3.
Fig. 3

Fig. 3. From: Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification.

Increase in Flk-H2B::YFP+ cells in BMP-2-induced tissue on days 2 and 4. Quantification of Flk1-H2B::YFP+ cells within the tissues 2 and 4 days after induction with Ad5-BMP-2-transduced or control cells. YFP+ nuclei were counted and reported as a ratio of the total area of the tissue section determined using the wide-field montage. Flk-H2B::YFP+ cells were significantly elevated in the tissues receiving BMP-2 compared with controls. The graph depicts the average number of Flk-H2B::YFP+ cells in five sections for day 2 control, 7 sections for day 2 BMP, 8 sections for day 4 control, and 6 sections for day 4 BMP. The number of images taken in each section ranged from 4 to 22. *Denotes a significant difference as determined by the Student's t test.

Christine Fouletier Dilling, et al. J Bone Miner Res. 2010 May;25(5):1147-1156.
4.
Fig. 2

Fig. 2. From: Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification.

Wide-field and confocal images of whole tissue sections and quantification of Flk1-H2B::YFP cells. (A, F) Representative montages of low-magnification gray-scale images (1 pixel = 0.003 mm) used for calculating total area for tissue sections. A single representative tissue section is depicted after the entire hind limb muscles that encompassed the injection site were isolated 2 days after receiving an intramuscular injection of cells transduced with either Ad5-empty control vector (A) or Ad5-BMP-2 (B) and sectioned at 15 µm thickness. Although every fifth section across the entire tissue was analyzed, we show only a single representative image of each type. The corresponding regions with positive YFP signal, shown by the boxed areas, were imaged by confocal microscopy (B–E, G–O) for counting the YFP+ cell numbers.

Christine Fouletier Dilling, et al. J Bone Miner Res. 2010 May;25(5):1147-1156.
5.
Fig. 5

Fig. 5. From: Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification.

Expression of VEGF-D during the early stages of endochondral bone formation. Results of qRT-PCR analysis of VEGF-A, -B, -C, and -D mRNA levels in tissues surrounding the lesional site that received either the Ad5-BMP-2- or Ad5-empty-transduced cells isolated at daily intervals for up to 7 days after initial injection. Four biologic replicates were run in triplicate, and the averages were normalized against an internal standard (ribosomal RNA). The samples receiving Ad5-BMP-2-transduced cells then were compared with those obtained from the tissues receiving cells transduced with Ad5-empty cassette virus. Therefore, the graph depicts the fold changes in VEGF RNAs in the BMP-2 samples over time compared with control tissues. Error bars depict ± 1 SD unit. *Denotes samples that had a statistically significant (p < .05) difference from all other samples by the ANOVA test.

Christine Fouletier Dilling, et al. J Bone Miner Res. 2010 May;25(5):1147-1156.
6.
Fig. 4

Fig. 4. From: Vessel Formation Is Induced Prior to the Appearance of Cartilage in BMP-2-Mediated Heterotopic Ossification.

Quantification of YFP+ cell proliferation. Representative images of Flk1-H2B::YFP and the cell proliferation marker Ki67. Colocalization of Flk1-H2B::YFP (yellow) and Ki67 (red) was detected in BMP-2-treated and control tissues. Graphs show the total number of YFP+/Ki67+ cells in the images taken within the area divided by the number of images analyzed. The area fraction was measured for nine at day 2 and five at day 4 BMP and eight at day 2 and four at day 4 control different areas, and the average area fraction was calculated for control and BMP-treated tissues. The area fractions of YFP+/Ki67+ nuclei in the control and the BMP-treated tissues on day 2 were 7.32 ± 3.26 and 10.20 ± 6.95, respectively. The area fractions for day 4 were 6.97 ± 2.32 and 11.26 ± 2.58 in the control and the BMP-treated tissues. Based on the Student's t test, the p value for the day 2 data was .29 and that for the day 4 data was .035. Taken together, the data showed significant YFP+/Ki67+ population increases by day 4 after the BMP treatment, but on day 2 there were no significant differences in dividing YFP cell population between control and BMP-treated tissues.

Christine Fouletier Dilling, et al. J Bone Miner Res. 2010 May;25(5):1147-1156.

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