Figure 1.
Biodistribution and homing of BMMCs.
(A) Representative coronal whole-body SPECT/CT images of control and silicosis animals 2, 4 and 24 h after endovenous 99mTc-BMMCs shows liver uptake until 24 h. n = 2 animals per group. 99mTc-BMMCs were found predominantly in the liver (arrows). (B) Real-time PCR analysis of GFP mRNA expressions. Adipose-derived mesenchymal stem cells from GFP mice were used as positive control (GFP Cells). Control (C) and silica group (SIL) mice received saline or silica intratracheally. C and SIL animals were treated with BMMC from GFP mice (C-Cell and SIL–cell) or saline (C-SAL). Data are presented as mean ± SEM. n = 4 animals per group. ***Significantly different from C-GFP (P<0.001). (C) Photomicrographs of spleen and lung parenchyma after imunohistochemistry with GFP antibody. GFP positive cells were not observed in lung parenchyma of C+BMMC and SIL+BMMC animals 24 h after treatment with BMMC from GFP mice. Spleen of GFP male mice were used as positive control. Bars: 100 µm. n = 4 animals per group.
Figure 2.
BMMC therapy improves lung function in SIL mice.
(A) Resistance (ΔP1,l) and viscoelastic (ΔP2,l) pressures and (B) lung static elastance (Est,l). Mice in the control (C) and silica (SIL) groups received saline or silica intratracheally. C and SIL animals were treated with BMMCs (1×106 cells i.v., C+BMMC and SIL+BMMC). Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from C. ##Significantly different from SIL40d and SIL70d.
Figure 3.
BMMC therapy increases cellularity in BALF, LDH activity and does not alter myeloperoxidase activity.
Quantification of (A) total number of cells, (B) mononuclear cells and (C) polymorphonuclear cells in BALF. Quantification of (D) myeloperoxidase and (E) LDH activity. Mice in the control (C) and silica (SIL) groups received saline or silica intratracheally. C and SIL animals were treated with BMMCs (1×106 cells i.v., C+BMMC and SIL+BMMC). Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from C. ##Significantly different from SIL40d and SIL70d.
Figure 4.
BMMC therapy does not reduce silicotic nodules.
(A) Photomicrographs of lung parenchyma stained with H&E from animals in the C, SIL40d, SIL70d and SIL+BMMC groups. Note the presence of silicotic nodules (*) containing lymphocytes (red arrow), neutrophils (green arrow) and macrophages (blue arrow) in the SIL groups. Bars: 100 µm. Quantification of (B) frequency, (C) nodules (>100,000), (D) volume (Vv) and (E) cellularity of silicotic nodules in the SIL groups. Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from SIL40d. ##Significantly different from SIL70d.
Figure 5.
BMMC therapy reduces silica particle inside nodules.
Photomicrographs of (A) silicotic nodules and (B) silica particle in nodules under polarization microscopy. Bars: 100 µm. (C) Quantification of silica inside nodules. Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from SIL40d. ##Significantly different from SIL70d.
Figure 6.
BMMC therapy decreases the presence of macrophages and cells reactive for iNOS.
Photomicrographs of lung parenchyma after histochemistry with (A) BSL-1 lectin and (B) iNOS antibody. Note the macrophages and cells reactive for iNOS (arrows) in the lung tissue. Bars: 100 µm. Quantification of BSL-1 macrophages in (C) septae and (D) nodules. (E) Quantification of iNOS cells in nodules. Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from SIL40d. ##Significantly different from SIL70d.
Figure 7.
BMMC therapy increases TGF-β expression and cell apoptosis.
Photomicrographs of lung parenchyma after immunohistochemistry with (A) TGF-β and (B) the TUNEL method. Note the apoptotic cells (arrows) in the silicotic nodules. Bars: 100 µm. Quantification of TGF-β in (C) septae and (D) nodules. (E) Quantification of apoptotic cells in nodules. Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from SIL40d. ##Significantly different from SIL70d.
Figure 8.
BMMC therapy leads to expansion and recruitment of T regulatory cells.
Photomicrographs of lung parenchyma after immunohistochemistry with FoxP3 antibody (A). Note the T regulatory cells (arrows) only in the SIL+BMMC group. Bars: 100 µm. Quantification of (B) TGF-β and (C) IL-10 by enzyme-linked immunosorbent assay. Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from C. ##Significantly different from SIL 40d. £Significantly different from SIL70d.
Figure 9.
BMMC therapy reduces collagen deposition.
(A) Photomicrographs of lung parenchyma stained with Sirius Red from animals in the C, SIL40d, SIL70d and SIL+BMMC groups. Bars: 100 µm. Quantification of collagen deposition on (B) septae and (C) nodules. Data are presented as the mean ± SEM. n = 7 animals per group. #Significantly different from C. ##Significantly different from SIL40d. £Significantly different from SIL70d.