Immunofluorescence analysis of microvascular density (MVD) and pericyte coverage of microvessels in the wound.

<p><b>(A)</b> A double-labeling technique was used to stain endothelial cells for CD31 expression (green) and pericytes for α-SMA expression (red). Representative images of the 2 groups at days 7 and 14 after the burn event are shown. Original magnification, ×200. <b>(B)</b> The MVD in the two groups at different time intervals. <b>(C)</b> The number of pericytes in the two groups at different time intervals. <b>(D)</b> The microvessel pericyte coverage index (MPI) was quantified by assessing the percentage of microvessels that were associated with α-SMA-positive pericytes. All data are expressed as the mean±SD (*P<0.05).</p

Immunofluorescence assay of proliferating endothelial cells in the wound.

<p><b>(A)</b> Representative examples of double staining of Ki67/CD31 (green, CD31; red, Ki67; nucleus, blue) in skin sections from the control group and the GM-CSF treatment group at day 3 after the burn event. Original magnification, ×200. (B) Quantitative comparison of the proliferating capillary index (PCI) in the two groups. The PCI was used to assess the percentage of microvessels with proliferating endothelial cells. All data are expressed as the mean±SD (*P<0.05).</p

Sequence of RT-PCR primers.

<p>Sequence of RT-PCR primers.</p

Effects of BM-MSC-derived conditioned medium samples on paracrine cell proliferation and migration.

<p>Equal numbers of keratinocytes, fibroblasts and HUVECs were incubated with vehicle control medium, norCM or hypoCM. Cell proliferation was evaluated at indicated time points (A, C and E). Data are given as the means±the SEM; *<i>p</i>< 0.05 compared with the vehicle control or the norCM group. #<i>p</i>< 0.05 the vehicle control compared with the norCM group. Equal numbers of keratinocytes, fibroblasts, HUVECs and CD14+ monocytes were added to the upper chambers of 24-well transwell plates, with the indicated medium added to the lower chambers (n = 4 wells per treatment). Cells that migrated to the bottom of the filter were stained and evaluated (B, D, F and G). Data are given as the means± the SEM;*<i>p</i>< 0.05 compared with the vehicle control or the norCM group. #<i>p</i>< 0.05 the vehicle control compared with the norCM group.</p

IHC evaluation of wounded mouse skin.

<p>Wound sections were evaluated on day 11 by staining with anti-Ki67 and anti-F4/80 antibodies. The numbers of Ki67+ proliferating cells (A, C) and recruited F4/80+ macrophages (B, D) in each of 4 randomly chosen high-power fields in the dermis were counted. Scale bar, 100 µm (400×). Data are expressed as the mean±the SEM;^*<i>p</i><0.05 compared with the vehicle control or the norCM group.</p

Electron micrographs of the newly formed microvessels in the wounds.

<p><b>(A)</b> Ultrastructural characterization of microvessels in the control group at day 14 after the burn event. The microvessels have a poorly organized basement membrane and swelled endothelial cells. Few pericytes coated the endothelium. Original magnification, ×5800. <b>(B)</b> In the GM-CSF treatment group, the endothelial cells formed tight junctions, several elongated pericytes were embedded within an integrated basement membrane, and their longitudinal processes completely surrounded the endothelial tube. E, endothelial cell; P, pericyte; BM, basement membrane. Original magnification, ×5800. <b>(C)</b> Enlargement of the image in the black box. A pericyte can be observed making direct contact with an endothelial cell. A high-power view of an EPI is shown, which is composed of a pericyte cytoplasmic projection and corresponding endothelial indentation. <b>(D)</b> Time course of vascular permeability changes after the burn event in the two groups. Data are expressed as the mean±SD (*P<0.05).</p

Proliferation of BM-MSCs in serum-containing complete culture medium (A) and serum-free α-MEM (B).

<p>Cell proliferation were examined under conditions of hypoxia or normoxia. Data are given as the means± the SEM; *<i>p</i>< 0.05 compared with the norCM group at indicated time points. #<i>p</i>< 0.05 compared with indicated earlier time points.</p

The speed and quality of burn wound healing.

<p><b>(A)</b> Photographs showing macroscopic wound healing in both the control and GM-CSF treatment groups at different time points following the burn event. <b>(B)</b> Wound healing in the control and GM-CSF treatment groups in self-controlled rat models at days 7 and 14 after the burn event. <b>(C)</b> Graph showing the wound healing rate in the two groups (mean±SD) (*P<0.05). <b>(D)</b> Histological changes in burn wounds in the two groups at days 14 and 21 after the burn event. Scale bar = 50 mm.</p

Expression of Ang-1 and Ang-2 in the skin during wound healing.

<p><b>(A)</b> Ang-1 mRNA levels in the control and GM-CSF treatment groups were assessed by qRT-PCR. <b>(B)</b> Immunohistochemical staining of Ang-1 protein in the two groups at day 7 after the burn event. Ang-1 protein signals were present mainly in pericyte-like perivascular mural cells. Scale bar = 100 mm. <b>(C)</b> Representative Western blots showing Ang-1 protein levels in the two groups during the healing process. <b>(D)</b> Statistical analysis of Ang-1 protein levels. <b>(E)</b> qRT-PCR analysis of Ang-2 mRNA expression levels in the two groups. <b>(F)</b> Immunohistochemical staining of Ang-2 protein at day 7 after the burn event. Ang-2 protein signals were present in capillary luminal and perivascular mural cells. Scale bar = 100 mm. <b>(G)</b> Representative Western blots showing Ang-2 protein levels during the healing process. <b>(H)</b> Statistical analysis of Ang-2 protein levels. <b>(I)</b> The protein expression ratio of Ang-1/Ang-2. All data above are expressed as the mean±SD (*P<0.05).</p

Increased capillary-like tube formation stimulated by BM-MSC hypoCM.

<p>(A) HUVECs were seeded onto a Matrigel matrix and incubated with vehicle control medium, hypoCM or norCM for 12 h. (B)Tube formation was quantified. Scale bar, 400 µm for images in (A) (200×). Results are given as the means ± the SEM; *<i>p</i>< 0.05 compared with the vehicle control or the norCM group.</p